Plasma derived from human umbilical cord blood for the treatment of neurodegenerative disorders

ABSTRACT

A method of treating neurodegenerative diseases using hUCB plasma is presented herein. hUCB plasma attenuated the hyperactive response (Group III) and potentiated the normal response in Group I ALS patients, but did not alter that of the non-responders to PHA (Group II). The elevated activity of caspase 3/7 observed in the MNCs from ALS patients was significantly reduced by hUCB plasma treatment. The ability of hUCB plasma to modulate the mitogen cell response and reduce caspase activity suggest that the use of hUCB plasma alone, or with stem cells, may prove useful as a therapeutic in ALS patients. hUCB plasma was shown to increase therapeutic efficacy of MNCs as well as decrease apoptosis of MNCs. The cytokine profile of hUCB plasma supports its usefulness as a sole therapeutic as well as an additive to MNCs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of and claims priority tocurrently pending U.S. Nonprovisional application Ser. No. 15/250,239,entitled “Plasma Derived from Human Umbilical Cord Blood for theTreatment of Neurodegenerative Disorders”, filed Aug. 29, 2016, whichclaims priority to U.S. Provisional Application No. 62/211,478, entitled“Plasma Derived from Human Umbilical Cord Blood for the Treatment ofNeurodegenerative Disorders”, filed Aug. 28, 2015, each of which isincorporated herein by reference.

FIELD OF INVENTION

This invention relates to treating neuronal diseases. Specifically, theinvention addresses treating neurodegenerative diseases, and/orneuro-inflammatory diseases using cord blood-derived plasma.

BACKGROUND OF THE INVENTION

Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a progressive degenerativedisease involving both upper and lower motor neuron damage in the spinalcord and brain. This disease clinically manifests as muscular weaknessand atrophy, which lead to paralysis and death of patients byrespiratory failure within 3 to 5 years (Rowland & Shneider, Amyotrophiclateral sclerosis. N. Engl. J. Med. 344(22):1688-1700; 2001). Most casesof ALS are sporadic; the familial (FALS), or genetically linked, form ofALS represents only 10 to 13 percent of all cases (Fiszman, et al.,Cu/Zn superoxide dismutase activity at different ages in sporadicamyotrophic lateral sclerosis. J. Neurol. Sci. 162(1):34-37; 1999;Pramatarova, et al., Identification of new mutations in the Cu/Znsuperoxide dismutase gene of patients with familial amyotrophic lateralsclerosis. Am. J. Hum. Genet. 56(3):592-596; 1995). About 20% of FALScases are the result of mutations in the gene for Cu/Zn superoxidedismutase (SOD1) that are associated with a decrease in SOD1 activity.Over 140 different SOD1 gene mutations have been reported (Andersen,Amyotrophic lateral sclerosis associated with mutations in the CuZnsuperoxide dismutase gene. Curr. Neurol. Neurosci. Rep. 6(1):37-46;2006). Available treatments for this disease lack the capacity to arrestdisease progression or repair motor neuron function. Cell therapy may bea promising new treatment for ALS.

Human umbilical cord blood (hUCB) may be preferable to other cellsources such as bone marrow due to hUCB cells' low pathogenicity andimmune immaturity. The mononuclear cell fraction from human hUCB (MNChUCB) is relatively rich in multipotent progenitors and has extensiveproliferation capacity (Mayani, & Lansdorp, Biology of human umbilicalcord blood-derived hematopoietic stem/progenitor cells. Stem Cells16(3):153-165; 1998; Todaro, et al., Hematopoietic progenitors fromumbilical cord blood. Blood Purif. 18(2):144-147; 2000). A number ofstudies have shown that intravenously administering MNC hUCB (Saneron'sproprietary fraction U-CORD-CELL™) into the jugular vein of G93A SOD1mice delayed the progression of disease and prolonged lifespan,increased motor neuron survival in the cervical/lumbar spinal cord,decreased pro-inflammatory cytokines (interleukin [IL]-1α, IL-1β, tumornecrosis factor [TNF]-α), and restored leukocyte profiles in these mice(Garbuzova-Davis, et al., Multiple intravenous administrations of humanumbilical cord blood cells benefit in a mouse model of ALS. PLoS One7(2):e31254; 2012; Garbuzova-Davis, et al., Human umbilical cord bloodtreatment in a mouse model of ALS: optimization of cell dose. PLoS One3(6):e2494; 2008; Garbuzova-Davis, et al., Intravenous administration ofhuman umbilical cord blood cells in a mouse model of amyotrophic lateralsclerosis: distribution, migration, and differentiation. J. Hematother.Stem Cell Res. 12(3):255-270; 2003). While multiple interdependentfactors may underlie the pathogenesis of ALS, increasing evidencesupports a role for autoimmune mechanisms (Alexianu, The role of immuneprocesses in amyotrophic lateral sclerosis pathogenesis. Rom. J. Neurol.Psychiatry 33(3-4):215-227; 1995; Appel, et al., Autoimmunity as anetiological factor in sporadic amyotrophic lateral sclerosis. Adv.Neurol. 68:47-57; 1995; Coban, et al., Serum anti-neuronal antibodies inamyotrophic lateral sclerosis. Int. J. Neurosci. 123(8):557-562; 2013;Niebroj-Dobosz, et al., Auto-antibodies against proteins of spinal cordcells in cerebrospinal fluid of patients with amyotrophic lateralsclerosis (ALS). Folia Neuropathol. 44(3):191-196; 2006; Pagani, et al.,Autoimmunity in amyotrophic lateral sclerosis: past and present. Neurol.Res. Int. 2011:497080; 2011). MNC hUCB were hypothesized to provideneuroprotective and/or trophic effects for motor neurons by modulatingthe host immune inflammatory system through release of various growth oranti-inflammatory factors. Additionally, hUCB plasma (hUCBP) is a richsource of cytokines and other proteins such as insulin-like growthfactor-1 (IGF-1), transforming growth factor (TGF)-β and vascularendothelial growth factor (VEGF) required for growth and survival ofhematopoietic stem cells (Broxmeyer, et al., Commentary: a rapidproliferation assay for unknown co-stimulating factors in cord bloodplasma possibly involved in enhancement of in vitro expansion andreplating capacity of human hematopoietic stem/progenitor cells. BloodCells 20(2-3):492-497; 1994; Kim, et al., Ex vivo expansion of humanumbilical cord blood-derived T-lymphocytes with homologous cord bloodplasma. Tohoku J. Exp. Med. 205(2):115-122; 2005; Lam, et al.,Preclinical ex vivo expansion of cord blood hematopoietic stem andprogenitor cells: duration of culture; the media, serum supplements, andgrowth factors used; and engraftment in NOD/SCID mice. Transfusion41(12):1567-1576; 2001). Moreover, it has been shown that hUCB serumcontains more neurotrophic factors (substance P, IGF-1, nerve growthfactor [NGF]) compared to the peripheral blood serum effectively usedfor the treatment of the persistent corneal epithelial defects(Vajpayee, et al., Evaluation of umbilical cord serum therapy forpersistent corneal epithelial defects. Br. J. Ophthalmol.87(11):1312-1316; 2003), neurotrophic keratitis (Yoon, et al.,Application of umbilical cord serum eyedrops for the treatment ofneurotrophic keratitis. Ophthalmology 114(9):1637-1642; 2007), andrecurrent corneal erosion (Yoon, et al., Application of umbilical cordserum eyedrops for recurrent corneal erosions. Cornea 30(7):744-748;2011). hUCBP has also been used as a replacement for fetal bovine serumin in vitro studies including the expansion of endothelial colonyforming cells (Huang, et al., Human umbilical cord blood plasma canreplace fetal bovine serum for in vitro expansion of functional humanendothelial colony-forming cells. Cytotherapy 13(6):712-721; 2011),mesenchymal stromal cells (MSCs) (Baba, et al., Osteogenic potential ofhuman umbilical cord-derived mesenchymal stromal cells cultured withumbilical cord blood-derived auto serum. J. Craniomaxillofac. Surg.40(8):768-772; 2012; Ding, et al., Human umbilical cord-derived MSCculture: the replacement of animal sera with human cord blood plasma. InVitro Cell. Dev. Biol. Anim. 49(10):771-777; 2013), T cells (Kim, etal., Ex vivo expansion of human umbilical cord blood-derivedT-lymphocytes with homologous cord blood plasma. Tohoku J. Exp. Med.205(2):115-122; 2005), and dental stem cells (Lee, et al., The effectsof platelet-rich plasma derived from human umbilical cord blood on theosteogenic differentiation of human dental stem cells. In Vitro Cell.Dev. Biol. Anim. 47(2): 157-164; 2011), demonstrating that it can exerta favorable influence on stem cells. These results suggest that hUCBPmay be effective as an additive to, or substitute for, cells indeveloping clinically useful protocols for cell-based ALS therapies.Including hUCBP with hUCB cells may add significant therapeutic benefitsand plasma alone may also be a useful treatment approach.

The inventors determined the efficacy of hUCBP on the functionalactivity of lymphocytes from the peripheral blood of ALS patients.First, hematological profiles were analyzed in the peripheral blood ofALS patients. Second, the mitogen-induced proliferation response of MNCsisolated from the peripheral blood of ALS patients in vitro whencultured with hUCBP were investigated. Finally, the effect of hUCBP uponthe apoptotic cell death response in ALS patients was examined.

Cord Blood Plasma

Cord blood plasma (CBP) is commonly obtained from human umbilical cordblood (hUCB) during cell isolation and has mainly been considered awaste product. However, the trophic effect of CBP has been shown inreplacing standard serum during the expansion of hUCB-derivedmesenchymal stem cells, human dental stem cells, hUCB-derivedT-lymphocytes, or human endothelial colony-forming cells in vitro. (DingY, Yang H, Feng J B, et al. Human umbilical cord-derived MSC culture:the replacement of animal sera with human cord blood plasma. In VitroCell Dev Biol Anim. 2013; 49:771-777; Lee J-Y, Nam H, Park Y-J, et al.The effects of platelet-rich plasma derived from human umbilical cordblood on the osteogenic differentiation of human dental stem cells. InVitro Cell Dev Biol Anim. 2011; 47:157-164; Kim Y-M, Jung M-H, Song H-Y,et al. Ex vivo expansion of human umbilical cord blood-derivedT-lymphocytes with homologous cord blood plasma. Tohoku J Exp Med. 2005;205:115-122; Huang L, Critser P J, Grimes B R, Yoder M C. Humanumbilical cord blood plasma can replace fetal bovine serum for in vitroexpansion of functional human endothelial colony-forming cells.Cytotherapy. 2011; 13:712-721). Moreover, the therapeutic potential ofCBP administration into rats modelling acute ischemic stroke wasdemonstrated by enhancement of neurogenesis and reduction ofinflammation leading to significant post-stroke functional recovery.(Yoo J, Kim H-S, Seo J-J, et al. Therapeutic effects of umbilical cordblood plasma in a rat model of acute ischemic stroke. Oncotarget. 2016;7:79131-79140). Also, tissue inhibitor of metalloproteinases, aplasticity-enhancing protein from CBP, has been found to promoterestoration of hippocampal function and memory in aged 18 months oldmice after CBP treatment. (Lee J-Y, Nam H, Park Y-J, et al. The effectsof platelet-rich plasma derived from human umbilical cord blood on theosteogenic differentiation of human dental stem cells. In Vitro Cell DevBiol Anim. 2011; 47:157-164; Castellano J M, Mosher K I, Abbey R J, etal. Human umbilical cord plasma proteins revitalize hippocampal functionin aged mice. Nature. 2017; 544:488-492).

A recent study showed beneficial functional improvement in anAlzheimer's disease (AD) mouse model by injection of a specific fractionfrom cord blood serum compared to adult blood serum. (Habib A, Hou H,Mori T, et al. Human umbilical cord blood serum-derived α-secretase:functional testing in Alzheimer's disease mouse models. Cell Transplant.2018). Additionally, umbilical cord serum has being effectively employedfor the treatment of corneal defects and neurotrophic keratitis inhumans. (Vajpayee R B, Mukerji N, Tandon R, et al. Evaluation ofumbilical cord serum therapy for persistent corneal epithelial defects.Br J Opthamol. 2003; 87:1312-1316; Yoon K-C, Choi W, You I-C, Choi J.Application of umbilical cord serum eyedrops for recurrent cornealerosions. Cornea. 2011; 30:744-748; Yoon K-C, You I-C, Im S-K, et al.Application of umbilical cord serum eyedrops for the treatment ofneurotrophic keratitis. Opthamol. 2007; 114:1637-1642).

In a relatively recent study, the inventors showed the ability of CBP tomodulate mitogen-induced in vitro proliferation of mononuclear cells(MNC) isolated from the peripheral blood of amyotrophic lateralsclerosis (ALS) patients. (Eve D J, Ehrhart J, Zesiewicz T, et al.Plasma Derived From Human Umbilical Cord Blood Modulates Mitogen-InducedProliferation of Mononuclear Cells Isolated From the Peripheral Blood ofALS Patients. Cell Transplant. 2016; 25:963-971). Interestingly, threedistinct cell responses to the mitogenic factor phytohemagglutinin werenoted, suggesting altered lymphocyte functionality in ALS patients. MNCresponses were shown to be regulated by CBP treatment in vitro.Additionally, the apoptotic activity of MNCs isolated from ALS patientswas significantly reduced by supplementing media with CBP. Thus, thesestudy results have not only broadened the therapeutic application of CBPfor ALS, but also further expanded its potential for treatment of otherneurodegenerative disorders with immunological aspects.

It has been shown that CBP contains high amounts of various growthfactors, such as vascular endothelial growth factor (VEGF), insulin-likegrowth factor-1 and transforming growth factor (TGF)-β, that arerequired for cell maintenance during hematopoiesis. (Kim Y-M, Jung M-H,Song H-Y, et al. Ex vivo expansion of human umbilical cord blood-derivedT-lymphocytes with homologous cord blood plasma. Tohoku J Exp Med. 2005;205:115-122; Lam A C, Li K, Zhang X B, et al. Preclinical ex vivoexpansion of cord blood hematopoietic stem and progenitor cells:duration of culture; the media, serum supplements, and growth factorsused; and engraftment in NOD/SCID mice. Transfusion. 2001;41:1567-1576). Although CBP can exert a favorable effect onhematopoietic stem cells, whether CBP elicits therapeutic benefit as anadditive to, or substitute for, cells must be determined beforedeveloping clinically relevant CBP-based therapies for variousneurodegenerative diseases.

The inventors characterized the composition of factors in CBP derivedfrom hUCB, which may mediate therapeutic benefit. First, cytokine andgrowth factor profiles were analyzed in the same CBP samples. Second,the efficacy of autologous CBP on MNC hUCB viability in vitro wasinvestigated. Finally, the effect of autologous CBP upon the apoptoticMNC hUCB response in vitro was examined. These study results provide abasis for further establishment of CBP as a self-contained therapeuticor as a supportive diluent for MNC hUCB infusion in treatment ofneurodegenerative diseases.

SUMMARY OF THE INVENTION

Treatment of a neuromotor degenerative disease is disclosed herein. Thetreatment comprises identifying a patient suffering from a neuromotordegenerative disease, such as through use of the ALS Functional RatingScale or ALS Functional Rating Scale or ALS Functional Rating Scale orALS Functional Rating Scale-revised methods. As such, in someembodiments, the neuromotor degenerative disease is amyotrophic lateralsclerosis. The patient is administered plasma derived from umbilicalcord blood. In specific variations of the invention the plasma derivedfrom umbilical cord blood is derived from human umbilical cord blood.

Other embodiments of the invention include the treatment of aneurodegenerative disease such as Alzheimer's disease, Parkinson'sdisease, multiple sclerosis, ischemia, or traumatic brain injury by theadministration of a therapeutically effective amount of human umbilicalcord blood plasma or alternatively, a therapeutically effective amountof human umbilical cord blood plasma in combination with atherapeutically effective amount of human umbilical cord blood cells. Insome embodiments, the human umbilical cord blood cells may be amononuclear fraction thereof.

Optionally, plasma derived from umbilical cord blood is administered atabout 10 ml/kg to about 20 ml/kg. As nonlimiting examples, the plasmaderived from umbilical cord blood can be administered at 9 ml/kg, 9.25ml/kg, 9.5 ml/kg, 9.75 ml/kg, 10 ml/kg, 10.25 ml/kg, 10.5 ml/kg, 10.75ml/kg, 11 ml/kg, 11.25 ml/kg, 11.5 ml/kg, 11.75 ml/kg, 12 ml/kg, 12.25ml/kg, 12.5 ml/kg, 12.75 ml/kg, 13 ml/kg, 13.25 ml/kg, 13.5 ml/kg, 13.75ml/kg, 14 ml/kg, 14.1 ml/kg, 14.2 ml/kg, 14.3 ml/kg, 14.4 ml/kg, 14.5ml/kg, 14.6 ml/kg, 114.7 ml/kg, 14.75 ml/kg, 14.8 ml/kg, 14.9 ml/kg, 15ml/kg, 15.1 ml/kg, 15.2 ml/kg, 15.25 ml/kg, 15.3 ml/kg, 15.4 ml/kg, 15.5ml/kg, 15.6 ml/kg, 15.7 ml/kg, 15.75 ml/kg, 15.8 ml/kg, 15.9 ml/kg, 16ml/kg, 16.1 ml/kg, 16.2 ml/kg, 16.25 ml/kg, 16.3 ml/kg, 16.4 ml/kg, 16.5ml/kg, 16.6 ml/kg, 16.7 ml/kg, 16.75 ml/kg, 16.8 ml/kg, 16.9 ml/kg, 17ml/kg, 17.25 ml/kg, 17.5 ml/kg, 17.75 ml/kg, 18 ml/kg, 18.25 ml/kg, 18.5ml/kg, 18.75 ml/kg, 19 ml/kg, 19.25 ml/kg, 19.5 ml/kg, 19.75 ml/kg, or20 ml/kg.

Optionally, a therapeutic composition is administered with the plasmaderived from umbilical cord blood. The therapeutic composition isriluzole, mesenchymal stem cells, umbilical cord blood cells, or acombination of the aforementioned compounds. In specific variations, thetherapeutic composition is umbilical cord blood cells, and may be amononuclear cell fraction of umbilical cord blood cells. In morespecific variations, the composition is a composition of CD34⁺ cellsfrom the umbilical cord blood cells.

In specific variations, the umbilical cord blood cells are administeredat about 1×10⁴ to about 5×10⁷ cells, about 1×10⁵ to about 9×10⁶ cells,about 2×10⁵ to about 8×10⁶ cells, or about 2×10⁵ cells. Nonlimitingexamples include 9×10³ cells, 1.0×10⁴ cells, 1.25×10⁴ cells, 1.5×10⁴cells, 1.75×10⁴ cells, 2.0×10⁴ cells, 2.25×10⁴, 2.5×10⁴ cells, 2.75×10⁴cells, 3.0×10⁴ cells, 3.25×10⁴ cells, 3.75×10⁴ cells, 4.0×10⁴ cells,4.25×10⁴ cells, 4.5×10⁴ cells, 4.75×10⁴ cells, 5.0×10⁴ cells, 5.25×10⁴cells, 5.5×10⁴ cells, 5.75×10⁴ cells, 6.0×10⁴ cells, 6.25×10⁴ cells,6.75×10⁴ cells, 7.0×10⁴ cells, 7.25×10⁴ cells, 7.75×10⁴ cells, 8.0×10⁴cells, 8.25×10⁴ cells, 8.75×10⁴ cells, 9.0×10⁴ cells, 9.25×10⁴ cells,9.75×10⁴ cells, 1.0×10⁵ cells, 1.25×10⁵ cells, 1.5×10⁵ cells, 1.75×10⁵cells, 2.0×10⁵ cells, 2.25×10⁵, 2.5×10⁵ cells, 2.75×10⁵ cells, 3.0×10⁵cells, 3.25×10⁵ cells, 3.75×10⁵ cells, 4.0×10⁵ cells, 4.25×10⁵ cells,4.5×10⁵ cells, 4.75×10⁵ cells, 5.0×10⁵ cells, 5.25×10⁵ cells, 5.5×10⁵cells, 5.75×10⁵ cells, 6.0×10⁵ cells, 6.25×10⁵ cells, 6.75×10⁵ cells,7.0×10⁵ cells, 7.25×10⁵ cells, 7.75×10⁵ cells, 8.0×10⁵ cells, 8.25×10⁵cells, 8.75×10⁵ cells, 9.0×10⁵ cells, 9.25×10⁵ cells, 9.75×10⁵ cells,1.0×10⁶ cells, 1.25×10⁶ cells, 1.5×10⁶ cells, 1.75×10⁶ cells, 2.0×10⁶cells, 2.25×10⁶, 2.5×10⁶ cells, 2.75×10⁶ cells, 3.0×10⁶ cells, 3.25×10⁶cells, 3.75×10⁶ cells, 4.0×10⁶ cells, 4.25×10⁶ cells, 4.5×10⁶ cells,4.75×10⁶ cells, 5.0×10⁶ cells, 5.25×10⁶ cells, 5.5×10⁶ cells, 5.75×10⁶cells, 6.0×10⁶ cells, 6.25×10⁶ cells, 6.75×10⁶ cells, 7.0×10⁶ cells,7.25×10⁶ cells, 7.75×10⁶ cells, 8.0×10⁶ cells, 8.25×10⁶ cells, 8.75×10⁶cells, and 9.0×10⁶ cells.

Alternatively, the umbilical cord blood cells are administered at about0.1×10⁶ cells/kg to about 10×10⁸ cells/kg, about 0.5×10⁶ cells/kg toabout 5×10⁸ cells/kg, or about 1×10⁷ cells/kg to about 2×10⁸ cells/kg.Nonlimiting examples include 1.0×10⁵ cells/kg, 1.25×10⁵ cells/kg,1.5×10⁵ cells/kg, 1.75×10⁵ cells/kg, 2.0×10⁵ cells/kg, 2.25×10⁵, 2.5×10⁵cells/kg, 2.75×10⁵ cells/kg, 3.0×10⁵ cells/kg, 3.25×10⁵ cells/kg,3.75×10⁵ cells/kg, 4.0×10⁵ cells/kg, 4.25×10⁵ cells/kg, 4.5×10⁵cells/kg, 4.75×10⁵ cells/kg, 5.0×10⁵ cells/kg, 5.25×10⁵ cells/kg,5.5×10⁵ cells/kg, 5.75×10⁵ cells/kg, 6.0×10⁵ cells/kg, 6.25×10⁵cells/kg, 6.75×10⁵ cells/kg, 7.0×10⁵ cells/kg, 7.25×10⁵ cells/kg,7.75×10⁵ cells/kg, 8.0×10⁵ cells/kg, 8.25×10⁵ cells/kg, 8.75×10⁵cells/kg, 9.0×10⁵ cells/kg, 9.25×10⁵ cells/kg, 9.75×10⁵ cells/kg,1.0×10⁶ cells/kg, 1.25×10⁶ cells/kg, 1.5×10⁶ cells/kg, 1.75×10⁶cells/kg, 2.0×10⁶ cells/kg, 2.25×10⁶, 2.5×10⁶ cells/kg, 2.75×10⁶cells/kg, 3.0×10⁶ cells/kg, 3.25×10⁶ cells/kg, 3.75×10⁶ cells/kg,4.0×10⁶ cells/kg, 4.25×10⁶ cells/kg, 4.5×10⁶ cells/kg, 4.75×10⁶cells/kg, 5.0×10⁶ cells/kg, 5.25×10⁶ cells/kg, 5.5×10⁶ cells/kg,5.75×10⁶ cells/kg, 6.0×10⁶ cells/kg, 6.25×10⁶ cells/kg, 6.75×10⁶cells/kg, 7.0×10⁶ cells/kg, 7.25×10⁶ cells/kg, 7.75×10⁶ cells/kg,8.0×10⁶ cells/kg, 8.25×10⁶ cells/kg, 8.75×10⁶ cells/kg, 9.0×10⁶cells/kg, 9.25×10⁶ cells/kg, 9.75×10⁶ cells/kg, 1.0×10⁷ cells/kg,1.25×10⁷ cells/kg, 1.5×10⁷ cells/kg, 1.75×10⁷ cells/kg, 2.0×10⁷cells/kg, 2.25×10⁷, 2.5×10⁷ cells/kg, 2.75×10⁷ cells/kg, 3.0×10⁷cells/kg, 3.25×10⁷ cells/kg, 3.75×10⁷ cells/kg, 3.8×10⁷ cells/kg,4.0×10⁷ cells/kg, 4.25×10⁷ cells/kg, 4.5×10⁷ cells/kg, 4.75×10⁷cells/kg, 5.0×10⁷ cells/kg, 5.25×10⁷ cells/kg, 5.5×10⁷ cells/kg,5.75×10⁷ cells/kg, 6.0×10⁷ cells/kg, 6.25×10⁷ cells/kg, 6.75×10⁷cells/kg, 7.0×10⁷ cells/kg, 7.25×10⁷ cells/kg, 7.75×10⁷ cells/kg,8.0×10⁷ cells/kg, 8.25×10⁷ cells/kg, 8.75×10⁷ cells/kg, 9.0×10⁷cells/kg, 9.25×10⁷ cells/kg, 9.75×10⁷ cells/kg, 1.0×10⁸ cells/kg,1.25×10⁸ cells/kg, 1.5×10⁸ cells/kg, 1.75×10⁸ cells/kg, 2.0×10⁸cells/kg, 2.25×10⁸, 2.5×10⁸ cells/kg, 2.75×10⁸ cells/kg, 3.0×10⁸cells/kg, 3.25×10⁸ cells/kg, 3.75×10⁸ cells/kg, 4.0×10⁸ cells/kg,4.25×10⁸ cells/kg, 4.5×10⁸ cells/kg, 4.75×10⁸ cells/kg, 5.0×10⁸cells/kg, 5.25×10⁸ cells/kg, 5.5×10⁸ cells/kg, 5.75×10⁸ cells/kg,6.0×10⁸ cells/kg, 6.25×10⁸ cells/kg, 6.75×10⁸ cells/kg, 7.0×10⁸cells/kg, 7.25×10⁸ cells/kg, 7.75×10⁸ cells/kg, 8.0×10⁸ cells/kg,8.25×10⁸ cells/kg, 8.75×10⁸ cells/kg, 9.0×10⁸ cells/kg, 9.25×10⁸cells/kg, 9.75×10⁸ cells/kg, and 1.0×10⁹ cells/kg.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1(A) is a graph showing the cytokine profile was assayed on plasmaderived from human umbilical cord blood and human adult serum using anultrasensitive human cytokine panel for IL-1β. Results are plotted asmean±SEM. Statistical significance was determined using two tailedt-tests (* p<0.001).

FIG. 1(B) is a graph showing the cytokine profile was assayed on plasmaderived from human umbilical cord blood and human adult serum using anultrasensitive human cytokine panel for IL-10. Results are plotted asmean±SEM. Statistical significance was determined using two tailedt-tests (* p<0.001).

FIG. 1(C) is a graph showing the cytokine profile was assayed on plasmaderived from human umbilical cord blood and human adult serum using anultrasensitive human cytokine panel for IL-6. Results are plotted asmean±SEM. Statistical significance was determined using two tailedt-tests (* p<0.001).

FIG. 1(D) is a graph showing the cytokine profile was assayed on plasmaderived from human umbilical cord blood and human adult serum using anultrasensitive human cytokine panel GM-CSF. Results are plotted asmean±SEM. Statistical significance was determined using two tailedt-tests (* p<0.001).

FIG. 1(E) is a graph showing the cytokine profile was assayed on plasmaderived from human umbilical cord blood and human adult serum using anultrasensitive human cytokine panel for IL-5. Results are plotted asmean±SEM. Statistical significance was determined using two tailedt-tests (* p<0.001).

FIG. 1(F) is a graph showing the cytokine profile was assayed on plasmaderived from human umbilical cord blood and human adult serum using anultrasensitive human cytokine panel for IFN-γ. Results are plotted asmean±SEM. Statistical significance was determined using two tailedt-tests (* p<0.001).

FIG. 1(G) is a graph showing the cytokine profile was assayed on plasmaderived from human umbilical cord blood and human adult serum using anultrasensitive human cytokine panel for TNF-α. Results are plotted asmean±SEM. Statistical significance was determined using two tailedt-tests (* p<0.001).

FIG. 1(H) is a graph showing the cytokine profile was assayed on plasmaderived from human umbilical cord blood and human adult serum using anultrasensitive human cytokine panel fir IL-2. Results are plotted asmean±SEM. Statistical significance was determined using two tailedt-tests (* p<0.001).

FIG. 1(I) is a graph showing the cytokine profile was assayed on plasmaderived from human umbilical cord blood and human adult serum using anultrasensitive human cytokine panel for IL-4. Results are plotted asmean±SEM. Statistical significance was determined using two tailedt-tests (* p<0.001).

FIG. 1(J) is a graph showing the cytokine profile was assayed on plasmaderived from human umbilical cord blood and human adult serum using anultrasensitive human cytokine panel for IL-8. Results are plotted asmean±SEM. Statistical significance was determined using two tailedt-tests (* p<0.001).

FIG. 2 is a graph showing hematological analysis of the peripheralblood. Seven ALS patients (Group I) had significantly (p=0.0278) lownormal WBC counts and two ALS patients (Group II) had higher counts thanhealthy volunteers. Although, there were no significant differences inRBC counts or hemoglobin level between ALS patients and healthyvolunteers, two patients from Group I had low normal RBC (3.9×106/μL and3.6×106/μL) and hemoglobin level (12.4 g/dL and 12.1 g/dL) compared toreference range for RBC (4.2-5.8×106/μL) and hemoglobin (13.2-17.1g/dL). Significantly fewer lymphocytes (p=0.0255) and elevatedneutrophils (p=0.0218) were noted in Group II compared to both Group Iand healthy volunteers.

FIG. 3 shows hematological analysis of the peripheral blood. Nosignificant differences in the hematological analysis of the peripheralblood were observed between Amyotrophic lateral sclerosis (ALS) patients(n=10) and healthy volunteers (n=5), except for a significant increasein monocyte number (*p<0.05).

FIG. 4 is a graph showing immunological analysis of the peripheralblood. Levels of IgG were significantly higher in Group I compared toboth healthy volunteers (p=0.0364) and Group II (p=0.0511), while theIgM profile was opposite, with significant (p=0.0357) elevation in GroupII. Note: Reference ranges for adults: IgG is 654-1618 mg/dL; IgM is48-271 mg/dL. Reference ranges for cord blood: IgG is 553-1360 mg/dL;IgM is <17 mg/dL

FIG. 5(A) is a microscopic image showing immunocytochemical analysis ofCD4. Scale bar in images is 25 μm.

FIG. 5(B) is a microscopic image showing immunocytochemical analysis ofCD8. Scale bar in images is 25 μm.

FIG. 6 is a graph showing PHA-induced proliferation of MNCs isolatedfrom peripheral blood in Medium 1 (containing FBS) and Medium 2(containing hUCBP). The response profile of mononuclear cells (MNCs)from healthy controls (n=5) to phytohemagglutinin (PHA; 10 μg/mL)stimulation when the cells were incubated with Medium 1 (fetal bovineserum [FBS] only containing) showed a normal increasing index ofstimulation (IS) with time. However, in ALS patients (n=12), this wasnot observed. A smaller, but similar effect was seen with the lower dose(1 μg/mL). The 10 μg/mL PHA IS was significantly higher than the 1 μg/mLat all time points for both ALS and controls (p<0.05) and the 72 hr 10μg/mL was significantly higher in controls.

FIG. 7 is a graph showing PHA-induced proliferation of MNCs isolatedfrom peripheral blood in Medium 1 (containing FBS) and Medium 2(containing hUCBP). Examination of the responses to PHA stimulationrevealed that there were three different response profiles for the ALSpatients' cells. The IS of MNCs from some ALS patients was similar(Group I; n=5), but abnormal extensive proliferation (increasedstimulation with a decreasing trend over time; Group III; n=1) andnon-inducible proliferation were also observed (Group II). Group II(n=6) was significantly different from both Group I and controls at bothconcentrations (* p<0.05) and the 10 μg/mL PHA IS was significantlyhigher than the 1 μg/mL at all time points for Group II ALS and controlonly (p<0.05).

FIG. 8 is a graph showing PHA-induced proliferation of MNCs isolatedfrom peripheral blood in Medium 1 (containing FBS) and Medium 2(containing hUCBP). When MNCs were cultured in Medium 2 containing hUCBplasma, the proliferation response of cells to PHA (10 μg/mL) of ALSpatients remained significantly reduced compared to controls (* p<0.05).

FIG. 9(A) is a microscope image showing PHA-induced proliferation ofMNCs isolated from peripheral blood of an ALS patient from Group III, inMedium 1 (containing FBS). Group III; abnormal extensive cellproliferation. Scale bar is 100 m.

FIG. 9(B) is a microscope image showing PHA-induced proliferation ofMNCs isolated from peripheral blood from a healthy control individual,in Medium 1 (containing FBS). Scale bar is 100 μm.

FIG. 9(C) is a microscope image showing PHA-induced proliferation ofMNCs isolated from peripheral blood of an ALS patient from Group III, inMedium 2 (containing hUCBP). Images show decreased numbers of coloniesin Medium 2 (Group III; abnormal extensive cell proliferation). Scalebar is 100 μm.

FIG. 9(D) is a microscope image showing PHA-induced proliferation ofMNCs isolated from peripheral blood from a healthy control individual,in Medium 2 (containing hUCBP). Images show decreased numbers ofcolonies in Medium 2. Scale bar is 100 μm.

FIG. 10 is a graph showing PHA-induced proliferation of MNCs isolatedfrom peripheral blood in Medium 1 (containing FBS) and Medium 2(containing hUCBP). Splitting the ALS patients into the previous 3groups based on their response to PHA in media 1, demonstrated that theproliferation response of cells to PHA (10 μg/mL) was blunted in cellsexhibiting abnormal extensive proliferation (Group III) when cultured inMedium 1. An insignificant increase in cell proliferation was observedin cultures with a “normal” response to PHA (Group I) and no significantdifferences between Media 1 and Media 2 were found in cell cultures withnon-inducible proliferation (Group II). Group II remained significantlydifferent from control and Group I with Medium 2 (* p<0.05).

FIG. 11 is a graph showing caspase 3/7 activity in MNCs isolated fromthe peripheral blood of ALS patients. Many Caspase-3/7-positive cellswere found in the MNCs of ALS patients cultured for 5 days in Medium 1,which was significantly different from that in controls (* p<0.05). WhenMedium 1 was changed to Medium 2 containing hUCB plasma for 24 hrs, theapoptotic activity of cells in the ALS patients was significantly lowerthan ion medium 1 (p<0.05).

FIG. 12 is a graph showing caspase 3/7 activity in MNCs isolated fromthe peripheral blood of ALS patients. More Caspase-3/7-positive cellswere found in patients with abnormal extensive proliferation (Group III)and non-inducible proliferation (Group II) compared to patients with“normal” response to PHA (Group I), though this was not significant.Cultured MNCs in Medium 2 showed significantly decreased apoptoticactivity in patients with an abnormal response to PHA stimulation(p<0.05).

FIG. 13(A) is a microscopic image showing caspase 3/7 activity in MNCsisolated from the peripheral blood of ALS patients. Images show thenumbers of Caspase 3/7 positive cells in Medium 1 (Group III) (red,asterisks). The nuclei are stained with Hoechst. Magnification is 20×.

FIG. 13(B) is a microscopic image showing caspase 3/7 activity in MNCsisolated from the peripheral blood of ALS patients. Images show thedecreased numbers of Caspase 3/7 positive cells in Medium 2 (Group III)(red, asterisks). The nuclei are stained with Hoechst. Magnification is20×.

FIG. 14 is a graph showing cord blood plasma decreases cell death invitro. Human umbilical cord blood cells were cultured in mediasupplemented with either cord blood plasma (CB Plasma), adult humanserum (HS) or fetal bovine serum (FBS). Cells cultured in cord bloodplasma demonstrated significantly greater live (dark gray) to dead(light gray) cells, compared to other groups using a Live/Dead viabilityassay kit. Cord blood plasma provided a beneficial environment that notonly supported cell survival with greater viability. Results are plottedas mean±SEM. Statistical significance was determined using two tailedt-tests (* p<0.001).

FIG. 15 is a graph showing cord blood plasma decreases cell death invitro. Human umbilical cord blood cells were cultured in mediasupplemented with either cord blood plasma (CB Plasma), adult humanserum (HS) or fetal bovine serum (FBS). Viability of cells in CB plasmasupplemented media was better in comparison to cultures supplementedwith either HS or FBS. Cord blood plasma provided a beneficialenvironment that not only supported cell survival with greaterviability.

FIG. 16A-J is a series of graphs depicting cord blood plasma cytokineprofile. The cytokine profiles of CBP (n=20) and ABP/S (n=6) wereassayed using an ultrasensitive human cytokine panel in triplicate.Significantly lower concentrations of the pro-inflammatory cytokines (B)IL-2, (E) IL-6, (H) IFN-γ and (I) TNF-α were detected in CBP vs. ABP/S.Levels of immunomodulatory (D) IL-5 cytokine and (J) GM-CSF weresignificantly low in CBP. A significant increase in (F) IL-8 was alsodetermined in CBP. There were no significant differences between CBP andABP/S for (A) IL-1β, (C) IL-4 and (G) IL-10. **P<0.01

FIG. 17 is a table (Table 1) depicting the levels of cytokines andgrowth factors presented as mean±SEM. CBP: Cord Blood Plasma; ABP/S:Adult Blood Plasma/Serum; Interleukin (IL): 1β, 2, 4, 5, 6, 8, and 10;IFN-γ: Interferon-gamma; TNF-α: Tumor necrosis factor-alpha; GM-CSF:Granulocyte-macrophage colony stimulating factor; VEGF: Vascularendothelial growth factor; G-CSF: Granulocyte-colony stimulating factor,EGF: Epithelial growth factor; FGF Basic: Fibroblast growth factorbasic. Significance of CBP vs. ABP/S denoted by: *P<0.05; **P<0.01.

FIG. 18A-D is a series of graphs depicting cord blood plasma growthfactor profile. The levels of the growth factors were analyzed in CBP(n=20) and ABP/S (n=6) using a human growth factor multiplex assay intriplicate. Significantly higher concentrations of (A) VEGF, (B) G-CSF,(C) EGF and (D) FGF basic growth factors were detected in CBP vs. ABP/S.*P<0.05, **P<0.01

FIG. 19A-B are a series of images depicting viability of MNC hUCB invitro. MNC hUCB (n=4 units) was cultured for 5 d in media supplementedwith either autologous CBP, ABP/S, or FBS in duplicate. The cells werestained using the LIVE/DEAD Viability/Cytotoxicity assay to identify theviable (dark grey fluorescence) and non-viable cytotoxic (light greyfluorescence) cell populations from images totaling n=16-20/supplementalcondition. (A) Confocal microscopy images demonstrated numerous viable(dark grey) MNC hUCB cultured with (Aa) CBP and (Ac) FBS supplements.Fewer viable cells were detected in culture supplemented with (Ab)ABP/S. Scale bar in Aa-Ac is 100 m. (B) MNCs cultured with autologousCBP supplement showed significantly greater cell survival vs. ABP/S.Also, media supplemented with CBP showed significantly reduced numbersof dead (light grey) MNC hUCB compared to FBS. Cells supplemented inmedia with CBP had a greater live (dark grey)/dead (light grey) cellratio compared to cultures that received ABP/S or FBS.*P<0.05, **P<0.01

FIG. 20A-B is a series of images depicting apoptotic activity of MNChUCB in vitro. MNC hUCB (n=6 units) was cultured for 5 d in mediasupplemented with either autologous CBP, ABP/S, or FBS in duplicate.Apoptosis was detected by TUNEL assay. (A) MNCs cultured in autologousCBP showed a significantly lower percentage of apoptotic absorbance vs.cultures supplemented with ABP/S or FBS. Cells incubated with FBS alsoexhibited significantly lower absorbance of apoptotic activity comparedto ABP/S. *P<0.05, **P<0.01, ***P<0.001. (B) Phase contrast images ofMNC hUCB in vitro demonstrated a few cells with abnormal morphologydisplaying dislocated nuclei in cultures supplemented with (Ba) CBPcompared to numerous morphologically damaged cells cultured with (Bb)ABP/S or (Bc) FBS, supporting apoptotic cell counts. Arrowheads indicatehealthy cells with normal morphology. Arrows indicate cells withabnormal morphology. Scale bar in Ba-Bc is 50 μm

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are described herein. All publicationsmentioned herein are incorporated herein by reference in their entiretyto disclose and describe the methods and/or materials in connection withwhich the publications are cited. It is understood that the presentdisclosure supersedes any disclosure of an incorporated publication tothe extent there is a contradiction.

All numerical designations, such as pH, temperature, time,concentration, and molecular weight, including ranges, areapproximations which are varied up or down by increments of 1.0 or 0.1,as appropriate. It is to be understood, even if it is not alwaysexplicitly stated that all numerical designations are preceded by theterm “about”. It is also to be understood, even if it is not alwaysexplicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art and can besubstituted for the reagents explicitly stated herein.

As used herein, the term “comprising” is intended to mean that theproducts, compositions and methods include the referenced components orsteps, but not excluding others. “Consisting essentially of” when usedto define products, compositions and methods, shall mean excluding othercomponents or steps of any essential significance. Thus, a compositionconsisting essentially of the recited components would not exclude tracecontaminants and pharmaceutically acceptable carriers. “Consisting of”shall mean excluding more than trace elements of other components orsteps.

As used herein, “about” means approximately or nearly and in the contextof a numerical value or range set forth means±15% of the numerical.

As used herein, “treat”, “treatment”, “treating”, and the like refer toacting upon a condition, such as autoimmune disease or immunotolerance,with an agent depending on the desired effect, to affect the conditionby improving or altering it. The improvement or alteration may includean improvement in symptoms or an alteration in the physiologic pathwaysassociated with the condition. “Treatment,” as used herein, covers oneor more treatments of a condition in a host (e.g., a mammal, typically ahuman or non-human animal of veterinary interest), and includes: (a)reducing the risk of occurrence of the condition in a subject determinedto be predisposed to the condition but not yet diagnosed, (b) impedingthe development of the condition, and/or (c) relieving the condition,e.g., causing regression of the condition and/or relieving one or morecondition symptoms (e.g., reduce inflammation).

As used herein, the terms “prophylactically treat” or “prophylacticallytreating” refers completely or partially preventing (e.g., about 50% ormore, about 60% or more, about 70% or more, about 80% or more, about 90%or more, about 95% or more, or about 99% or more) a condition or symptomthereof and/or may be therapeutic in terms of a partial or complete cureor alleviation for a condition and/or adverse effect attributable to thecondition.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptablediluent,” “pharmaceutically acceptable carrier,” or “pharmaceuticallyacceptable adjuvant” means an excipient, diluent, carrier, and/oradjuvant that are useful in preparing a pharmaceutical composition thatare generally safe, non-toxic and neither biologically nor otherwiseundesirable, and include an excipient, diluent, carrier, and adjuvantthat are acceptable for veterinary use and/or human pharmaceutical use.“A pharmaceutically acceptable excipient, diluent, carrier and/oradjuvant” as used in the specification and claims includes one or moresuch excipients, diluents, carriers, and adjuvants.

The term “therapeutically effective amount” as used herein describesconcentrations or amounts of components such as stem cells, plasma orother agents which are effective for producing an intended result,including preventing further neurodegenerative disease, or treating aneurodegenerative disease, such as ischemia, amyotrophic lateralsclerosis, Alzheimer's disease, traumatic brain injury, Parkinson'sdisease or multiple sclerosis. Compositions according to the presentinvention may be used to effect a favorable change on immune or neuronalcells, whether that change is an improvement, such as stopping orreversing the disease, or relieving to some extent one or more of thesymptoms of the condition being treated, and/or that amount that willprevent, to some extent, one or more of the symptoms of the conditionthat the host being treated has or is at risk of developing, or acomplete cure of the disease or condition treated.

The term “administration” refers to introducing an agent of the presentdisclosure into a patient. One preferred route of administration of theagent is oral administration. Another preferred route is intravenousadministration. However, any route of administration, such asparenteral, topical, subcutaneous, peritoneal, intraarterial,inhalation, vaginal, rectal, nasal, introduction into the cerebrospinalfluid, or instillation into body compartments can be used.

As used herein, the term “subject,” “patient,” or “organism” includeshumans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses).Typical patients to which an agent(s) of the present disclosure may beadministered will be mammals, particularly primates, especially humans.For veterinary applications, a wide variety of subjects will besuitable, e.g., livestock such as cattle, sheep, goats, cows, swine, andthe like; poultry such as chickens, ducks, geese, turkeys, and the like;and domesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals will besuitable subjects, including rodents (e.g., mice, rats, hamsters),rabbits, primates, and swine such as inbred pigs and the like.

The term “umbilical cord blood” is used herein to refer to bloodobtained from a neonate or fetus, most preferably a neonate andpreferably refers to blood that is obtained from the umbilical cord orthe placenta of newborns. Preferably, the umbilical cord blood isisolated from a human newborn. The use of umbilical cord blood as asource of mononuclear cells is advantageous because it can be obtainedrelatively easily and without trauma to the donor. Umbilical cord bloodcells can be used for autologous transplantation or allogenictransplantation, when and if needed. Umbilical cord blood is preferablyobtained by direct drainage from the cord an/or by needle aspirationfrom the delivered placenta at the root and at distended veins. As usedherein, the term “cells obtained from umbilical cord blood” refers tocells that are present within umbilical cord blood. In one embodiment,the cells obtained from umbilical cord blood are mononucleated cellsthat are further isolated from the umbilical cord blood.

Example 1

The human umbilical cord blood plasma (hUCBP) was obtained duringisolation of the MNC hUCB (Saneron CCEL Therapeutics Inc.; n=4; 1 male:3 female). The blood was collected in sterile tubes with heparin (10units of heparin per 1 mL of blood; BD, Franklin Lakes, N.J., USA) atthe time of birth using venipuncture of the umbilical vein. The UCB wasdiluted (1:1) with sterile phosphate buffered saline (PBS) without Mg²⁺and Ca²⁺ (Sigma-Aldrich, St. Louis, Mo., USA) at 37° C., and overlaid on12.5 mL of Ficoll (Ficoll-Paque™ Premium 1.077, GE Healthcare, Cat No.17-5442-02) in 50 mL sterile centrifuge tubes (BD Falcon, Cat No.352074, Bedford, Mass., USA). The blood samples were centrifuged at400×g for 40 min at 26° C. and the mononuclear cell (MNC) layer wastransferred with plasma to new 50 mL tubes by using 10 mL serologicalpipettes (Fisher brand, Cat No. 13-678-11E, Waltham, Mass., USA). TheMNCs with plasma were centrifuged at 440×g for 30 min at 21° C. and theplasma collected from the tube. Plasma was stored at −20° C. The MNCswere washed twice in 30 mL of PBS at 440×g for 13 min at 21° C. The cellnumbers and viability were determined using a Vi-CELL Viability Analyzer(Beckman Coulter, Brea, Calif., USA). The MNCs were frozen inCryopreservation Media (Saneron CCEL Therapeutics, Inc. Tampa, Fla.,USA) at 2×10⁶ cells per vial and stored in liquid nitrogen.

Data are presented as mean±S.E.M. The results were evaluated using ANOVAand Tukey's post hoc test or a paired Student's t-test (Excel;Microsoft, Redmond, Wash., USA). A p value<0.05 was consideredsignificant.

A cytokine profile was performed on the cord blood plasma compared tocommercially available adult human serum (Atlanta Biologicals, Cat. No.540110). Cord blood plasma was found to possess higher levels of thepro- and immunomodulatory cytokines IL-1β and IL-8 compared to adultblood serum, as seen in FIG. 1(A) and FIG. 1(J). However, the cord bloodpossessed lower levels of IL-10, IL-6, GM-CSF, IL-5, IFN-γ, TNF-α, IL-2,and IL-4 compared to adult blood serum, as seen in FIGS. 1(B), 1(C),1(D), 1(E), 1(F), 1(G), 1(H), and 1(I). The cytokine levels weresignificantly different for IL-5, IFN-γ, TNF-α, IL-2, IL-4, GM-CSF, andIL-6, major pro-inflammatory cytokines. This evidences theanti-inflammatory and immuno-modulatory properties of cord blood plasma.As such, cord blood plasma is a useful therapeutic agent, and canalternatively be used as a diluent in cell administration in order toprovide a beneficial environment for the transplanted cells.

Example 2

A total of twelve ALS patients (11 males and 1 female, mean age 53±2.7years; range 39-69), with a confirmed diagnosis of “Definite ALS” by aBoard-certified neurologist (primary neurologist), and six healthyvolunteers (3 males and 3 females, mean age 61.3±4.8 years; range 38-69)were enrolled in the study, as seen in the Table. Eleven patients wereCaucasian, and one patient was African-American. The healthy volunteerswere gender- and age-matched to ALS patients and had no neurological,autoimmune, systemic, or psychiatric diseases. Each participant signedan Informed Consent Form prior to enrolling in the study. The PatientCare Database Form and Medical History Form were completed by eachpatient and healthy volunteer. A neurological exam was performed uponeach study participant. Each study participant was graded on the ALSFunctional Rating Scale (ALSFRS; maximum score 40) and ALSFRS-revised(ALSFRS-R; including pulmonary/respiratory function; maximum score 48)using the on-line ALS C.A.R.E. Program (Center for Outcomes Research,Univ. Massachusetts Medical School, Worcester, Mass.) from datacollected by the same neurologist.

The ALS patients were divided into three groups based on their ALSFRSassessment scores with four patients in each; Group 1 (late stage;ALSFRS<20; 17.75±0.9), Group 2 (intermediate; 20<ALSFRS<30; 22±0.7), andGroup 3 (early stage; ALSFRS score>30; 32.5±1.0). The three groups aresignificantly different based on ALSFRS and ALSFRS-R scores (p<0.05) butnot age, disease duration or time from diagnosis. All healthy controlpatients scored 40/48 on the ALSFRS/ALSFRS-R assessments.

TABLE 1 ALS patient demographics. All ALS Patients grouped by ALSFRSScore patients Group 1 Group 2 Group 3 Healthy volunteers n 12 4 4 4 6Age (years)   53 ± 2.7 51.3 ± 6.4 54.0 ± 5.2 53.8 ± 3.1 61.3 ± 4.8 mean± SEM (39-69) (39-69) (39-63) (45-59) (38-69) Sex 11/1 4/0 3/1 4/0 3/3(male/female) ALSFRS 24.1 ± 1.9 17.8 ± 0.9   22 ± 0.7 32.5 ± 1.0 40.0 ±0.0 mean ± SEM (15-35) (15-19) (21-24) (30-35) ALSFRS-R 30.7 ± 2.1   24± 1.5 28.5 ± 1.6 39.5 ± 1.0 48.0 ± 0.0 mean ± SEM (21-41) (21-27)(25-32) (37-41) Disease onset 42.5 ± 7.8  53.5 ± 13.9   47 ± 16.8   27 ±8.4 NA (months) (11-96) (26-88) (20-96) (11-49) mean ± SEM Months since21.5 ± 4.6   26 ± 6.9 25.8 ± 9.9 12.8 ± 7.1 NA diagnosis  (5-53) (13-43) (7-53)  (5-34) mean ± SEM

Peripheral blood (˜80 mL) from ALS patients and healthy volunteers wasobtained via venipuncture by a nurse. Hematological analysis (completeblood cell [CBC] and white blood cell differential [WBCD] counts) wasperformed for each blood sample (performed by Quest Diagnostics, Inc.,Madison, N.J.). Data are presented as mean±S.E.M. The results wereevaluated using ANOVA and Tukey's post hoc test or a paired Student'st-test (Excel; Microsoft, Redmond, Wash., USA). A p value<0.05 wasconsidered significant.

Seven ALS patients (Group I) had significantly (p=0.0278) low normal WBCcounts and two ALS patients (Group II) had higher counts than healthyvolunteers. Although, there were no significant differences in RBCcounts or hemoglobin level between ALS patients and healthy volunteers,two patients from Group I had low normal RBC (3.9×10⁶/μL and 3.6×10⁶/μL)and hemoglobin level (12.4 g/dL and 12.1 g/dL) compared to referencerange for RBC (4.2-5.8×10⁶/μL) and hemoglobin (13.2-17.1 g/dL). However,in general Group I ALS patients exhibited lower WBC compared tocontrols, whereas Group II ALS patients exhibited higher WBC counts, asseen in FIG. 2 . An analysis of the WBC constituents showed controlpatient blood contains slightly higher lymphocyte cells and eosinophils,whereas ALS patient possess slightly higher neutrophil counts, andhigher monocyte cell counts, as seen in FIG. 3 . Of the differencesseen, only the alterations in monocyte levels were statisticallysignificant, which were significantly higher in all ALS patients (8.98%vs. 7.3%; p<0.05). However, when the ALS patient population wassegregated based on ALSFRS, significantly fewer lymphocytes (p=0.0255)and elevated neutrophils (p=0.0218) were noted in Group II compared toboth Group I and healthy volunteers.

Levels of IgG were significantly higher in Group I compared to bothhealthy volunteers (p=0.0364) and Group II (p=0.0511), while the IgMprofile was opposite, with significant (p=0.0357) elevation in Group II,seen in FIG. 4 . By comparison, typically ranges for IgG in healthyadults is 654-1618 mg/dL, and for IgM is 48-271 mg/dL. Additionally, thereference ranges for IgG in cord blood is 553-1360 mg/dL, and for IgM is<17 mg/dL.

Blood smears from each blood sample were fixed in methanol forimmunocytochemical analysis of CD4 and CD8 cells. Briefly, the mousemonoclonal antibodies CD4 (ab848) or CD8 (ab17147) (1:200, Abcam PLC,Cambridge, UK) were applied on a slide after 60 min pre-incubation with10% normal goat serum and Triton X100 in phosphate buffered saline(PBS). After incubating overnight at 4° C., the slides were washed andincubated with goat anti-mouse secondary antibody conjugated torhodamine (1:1200, Alexa, Molecular Probes) or FITC (1:500, Alexa,Molecular Probes) for 2 hrs at room temperature. Then the slides wererinsed in PBS and coverslipped with Vectashield (DAPI, Vector) andexamined under epifluorescence. Counts of CD4 and CD8 positive cellswere performed on five representative images from each slide usingImagePro Software. The percentages of CD4 and CD8 positive cells werecalculated based upon the total number of DAPI positive cells. Also,routine Giemsa staining was performed for each blood sample.

The ratio of CD4 staining, compared to CD8 staining was analyzed. InGroup I ALS patients, the ratio of CD4/CD8 was similar to healthycontrol blood samples (1.63±0.13 for Group I, 1.59±0.09 for control).However, in Group II ALS patients, the ratio of CD4/CD8 was elevated(1.86±0.11), as seen in FIG. 5 .

ALS patients showed hematological and immunological differencesdepending upon the stage of disease. Patients in Group I, as defined byALSRS, had significantly lower WBC counts and higher IgG levels thanpatients in Group II. The Group II patients had significantly higherpercentages of neutrophils and lower percentages of lymphocytes in WBC,higher IgM levels, and an elevated CD4/CD8 ratio. These results mayindicate early stage infections and/or inflammation in the Group IIpatients

Example 3

Fresh peripheral blood from ALS patients and healthy volunteers wascollected in sterile tubes with heparin (10 units of heparin per 1 mL ofblood; BD, Franklin Lakes, N.J., USA) and diluted (1:1) with sterilephosphate buffered saline (PBS) without Mg²⁺ and Ca²⁺ (Sigma-Aldrich,St. Louis, Mo., USA) at 37° C. Then, 12.5 mL of Ficoll (Histopaque-1077,Sigma-Aldrich, Cat No. 10771) was added into 50 mL sterile centrifugetubes (BD Falcon, Cat No. 352074, Bedford, Mass., USA). Blood samplesdiluted in PBS were overlaid on the Ficoll and centrifuged at 400×g for40 min at 26° C. The MNC layer was transferred with plasma to new 50 mLtubes by using 10 mL serological pipettes (Fisherbrand, Cat No.13-678-11E, Waltham, Mass., USA). The MNCs were washed twice in 30 mL ofPBS at 440×g for 13 min at 21° C. The cell numbers and viability weredetermined using a Vi-CELL Viability Analyzer (Beckman Coulter, Brea,Calif., USA). The MNCs were frozen in Cryopreservation Media (SaneronCCEL Therapeutics, Inc. Tampa, Fla., USA) at 2×10⁶ cells per vial andstored in liquid nitrogen. Cell samples contained approximately 7.4million white blood cells per millimeter, 11.6% granulocytes, and 1-4%CD34⁺ cells.

Cryopreserved MNCs were thawed rapidly at 37° C. then transferred slowlywith a pipette into a 15-ml centrifuge tube containing sterile PBS. Thecells were centrifuged (400×g/7 min), the supernatant discarded, and theprocess repeated. After the final wash, viability of cells was assessedusing the 0.4% trypan blue dye exclusion method prior to culture. Thecells (25×10³) were plated in triplicate in 96-well plates (FisherBrand) with Roswell Park Memorial Institute (RPMI)-1640/10% fetal bovineserum (FBS) (Medium 1; all from Sigma-Aldrich). After 24 hoursincubation, phytohemagglutinin (PHA; Sigma-Aldrich) was added to theculture at 1 μg/mL or 10 μg/mL. The cell colonies in the entire wellwere counted at 24, 48, and 72 hours incubation. The index ofstimulation (IS) was determined as the number of induced colonies/numberspontaneous colonies in the control wells.

Data are presented as mean±S.E.M. The results were evaluated using ANOVAand Tukey's post hoc test or a paired Student's t-test (Excel;Microsoft, Redmond, Wash., USA). A p value<0.05 was consideredsignificant.

The peripheral blood isolated MNCs were cultured in vitro with themitogen PHA, seen in FIG. 6 . There were three different responseprofiles of MNCs to PHA (10 g/mL) stimulation when the cells wereincubated with Medium 1. In healthy control volunteers, the index ofstimulation was 32 at 24 h to 50 at 72 h of incubation and showedapparent linear increases over time. MNCs from some ALS patients wassimilar, but abnormal extensive proliferation (increased stimulationwith a decreasing trend over time) and non-inducible proliferation wereobserved, from a value of 10 at 24 hours post-treatment to a value of 20at 72 hours post-treatment for the lower treatment dose (1 ag/mL PHA).Higher dosages (10 μg/mL PHA) display a similar relationship, withvalues ranging from 20 at 24 hours post-treatment to 40 at 72 hourspost-treatment. These trends display a typical time- and dose-dependentincrease in response to PHA stimulation (p<0.05; n=5). Dose-dependenteffects were seen in ALS patients, as the high-dose PHA stimulationconsistently increased the index of stimulation (p<0.05), but notime-dependent increases were observed. At 72 hrs and a dose of 10μg/ml, the control patients' IS was significantly higher than that forthe ALS MNCs. Interestingly, low response to PHA at 1 μg/mLconcentration was found in all ALS patients compared to control healthyvolunteers.

Further analysis of the ALS data revealed three distinct profiles thatemerged when the isolated MNCs were incubated with PHA, seen in FIG. 7 .The index of stimulation (IS) for some ALS patients was similar to thatof controls showing the typical time- and dose-dependent responsewithout significant difference (Group I; n=5). However, abnormalextensive proliferation (an increased stimulation with a decreasingtrend over time) was observed in one patient (Group III; this is neitherthe female patient, the African-American, nor the patient with thelowest ALS score, though it is the oldest patient). Non-inducibleproliferation was observed with MNCs isolated from other ALS patients(Group II; n=6). Group II showed a significant dose-dependent responseat each time point (p<0.05) and was significantly reduced compared toGroup I and controls at 48 and 72 hrs for both concentrations of PHA.Additionally, MNCs isolated from human umbilical cord blood (hUCB)showed little to no cell proliferation with either concentration of PHAused (data not shown). The normal, abnormal extensive proliferation andnon-responding patients did not correlate with the threeALSFRS-designated groups. Re-analysis of the previous parameters usingthis grouping also did not reveal any significant differences. SinceGroup III only contained one patient, no statistics could be performedusing this group.

ALS patients differed in lymphocyte functionality, possible due todifferences in immune response. Patients with abnormally extensive cellproliferation (Group III) in response to mitogen (PHA) in vitro probablyhave autoimmunity impairment while non-inducible proliferation (GroupII) may indicate immune deficiency.

Example 4

The hUCB plasma (hUCBP) was obtained during isolation of the MNC hUCBcells, as described previously. The blood was collected in sterile tubeswith heparin (10 units of heparin per 1 mL of blood; BD, Franklin Lakes,N.J., USA) at the time of birth using venipuncture of the umbilicalvein. The UCB was diluted (1:1) with sterile phosphate buffered saline(PBS) without Mg²⁺ and Ca²⁺ (Sigma-Aldrich, St. Louis, Mo., USA) at 37°C., and overlaid on 12.5 mL of Ficoll (Histopaque-1077, Sigma-Aldrich,Cat No. 10771) in 50 mL sterile centrifuge tubes (BD Falcon, Cat No.352074, Bedford, Mass., USA). The blood samples were centrifuged at400×g for 40 min at 26° C. and the mononuclear cell (MNC) layer wastransferred with plasma to new 50 mL tubes by using 10 mL serologicalpipettes (Fisherbrand, Cat No. 13-678-11E, Waltham, Mass., USA). Plasmawas stored at −20° C.

Peripheral blood (˜80 mL) from was obtained from the ALS patients andhealthy volunteer population via venipuncture by a nurse and processedas described in Example 1. Briefly, blood was collected in heparin tubes(BD, Franklin Lakes, N.J., USA) and diluted in PBS without Mg²⁺ and Ca²⁺(Sigma-Aldrich, St. Louis, Mo., USA) at 37° C., followed by Ficollextraction (Histopaque-1077, Sigma-Aldrich, Cat No. 10771). The MNCswere washed twice in 30 mL of PBS and MNCs were frozen inCryopreservation Media (Saneron CCEL Therapeutics, Inc. Tampa, Fla.,USA) at 2×10⁶ cells per vial and stored in liquid nitrogen.

Cryopreserved MNCs, described in Example 2, were thawed rapidly at 37°C. then transferred slowly with a pipette into a 15-ml centrifuge tubecontaining sterile PBS. The cells were centrifuged (400×g/7 min), thesupernatant discarded, and the process repeated. Cell viability wasdetermined using trypan blue dye and the cells (25×10³) plated intriplicate in 96-well plates (Fisher Brand) with RPMI-1640/10% fetalbovine serum (FBS) (Medium 1; all from Sigma-Aldrich), or RPMI-1640/10%hUCBP ABO Rh matched (Medium 2). After 24 hours incubation,phytohemagglutinin (PHA; Sigma-Aldrich) was added to the culture at 1μg/mL or 10 μg/mL. The cell colonies in the entire well were counted at24, 48, and 72 hours after addition of PHA. The index of stimulation(IS) was determined as the number of induced colonies/number spontaneouscolonies in the control wells.

Data are presented as mean±S.E.M. The results were evaluated using ANOVAand Tukey's post hoc test or a paired Student's t-test (Excel;Microsoft, Redmond, Wash., USA). A p value<0.05 was consideredsignificant.

Isolated MNCs cultured with media supplemented with plasma collectedfrom hUCB (Medium 2) and treated with PHA showed a non-significantincrease in the IS after incubating on Medium 2 at each point. In thehealthy control population, the difference in index of stimulationbetween media and plasma-supplemented media increased as timeprogressed, with a difference under 5 at 24 hours and about 10 by 72hour, as seen in FIG. 8 . Cells from all ALS patients appeared toexhibit a mild time-dependent IS response, which was significantly lowerthan that for the control MNCs at 48 and 72 hrs. However, segregatingthe ALS population based on PHA response, as undertaken in Example 3,revealed that stimulation of the cells that exhibited abnormal extensiveproliferation (Group III) using Medium 1 resulted in clustering of GroupIII cells, not seen in the control group, as seen in FIGS. 9(A) and9(B). By comparison, the UCB plasma-supplemented medium (Medium 2)showed a blunted expansion, as seen in FIG. 9(C), compared to thecontrol group seen in FIG. 9(D). The modulated stimulation effect seenwith Medium 2 was observed at all time points, as seen in FIG. 10 .Insignificant increases were observed in cultures with a standardresponse to PHA (Group I; n=5), while no differences between Medium 1and Medium 2 were observed from cell cultures that exhibitednon-inducible proliferation (Group II). Group II MNCs had asignificantly lower index of stimulation than Group I and controls atboth 48 and 72 hours with regards to Medium 2. Again, no differenceswere observed when the patients were grouped by ALSFRS and nocorrelations were evident.

ALS patients differed in lymphocyte functionality, possibly due todifferences in immune response. Patients with abnormally extensive cellproliferation (Group III) in response to mitogen (PHA) in vitro probablyhave autoimmunity impairment while non-inducible proliferation (GroupII) may indicate immune deficiency. Cord blood plasma modulates the cellresponse to the mitogen (PHA) only in patients with abnormally extensivecell proliferation and was not effective in patients with non-induciblecell proliferation.

These initial results demonstrate that plasma derived from cord bloodcould be effective in ALS patients with immune dysfunction.

Example 5

Caspase 3/7 activity was determined in MNCs isolated from the peripheralblood of ALS patients to determine the potential of these cells toundergo apoptosis. MNCs isolated from the peripheral blood of ALSpatients and healthy volunteers, as described in Example 2. The MNCswere plated and incubated in Medium 1 as described above for 5 days,after which the cells were incubated in Medium 2 for 24 hrs. Caspase 3/7activities were determined in these cells using a Magic Red Caspase 3/7kit (Immunochemistry Technologies, LLC, Bloomington, Minn., USA).Briefly, 10 μL of the 31× MagicRed-(aspartate-glutamate-valine-aspartate)², [MR-(DEVD)²] solution wasadded to each cell well and incubated for 1 hour. Hoechst dye (nucleistaining; Sigma-Aldrich) was added at 1 μL/well and incubated for anadditional 5 min. Immediately after incubation, five representativephotomicrographs were produced and counts of Caspase 3/7- andHoechst-positive cells were performed using ImagePro Software. ApoptoticCaspase 3/7 cells were expressed as the percentage of the total Hoechstcells.

Data are presented as mean±S.E.M. The results were evaluated using ANOVAand Tukey's post hoc test or a paired Student's t-test (Excel;Microsoft, Redmond, Wash., USA). A p value<0.05 was consideredsignificant.

MNCs, isolated from ALS patients, cultured in medium 1 showed manycaspase 3/7-positive cells with significantly more pronounced expressionin cells compared to controls (p<0.0⁵), as seen in FIG. 11 . Theincrease in caspase-3 and 7-positive cells was more pronounced in inpatients with abnormal extensive proliferation (7.38%, Group III), andnon-inducible proliferation (5.81%, Group II), compared to patients with“normal” response to PHA (4.58%, Group I) or MNC hUCB (3.75%). Caspaseactivity of the ALS patients generally showed more activity in patientsthat exhibited abnormal extensive proliferation or non-inducibleproliferation compared to MNCs that showed a normal response to PHA.Using Medium 2 supplemented with hUCB plasma resulted in significantlylower apoptotic activity after a 24-hour incubation for all ALS(p<0.05). However, group analysis suggested that only the Group 1(ALSFRS<20) and Group 3 (ALSFRS>30) patients had significantly reducedlevels of caspase 3/7 (p<0.05; data not shown). When grouped by theirresponse to PHA, only MNCs from patients that exhibited an abnormalresponse to PHA stimulation, i.e. Groups II and III, showed decreasedapoptotic activity (p<0.05) when cultured in Medium 2, as seen in FIG.12 . Images of the stained cells show higher numbers of cells stainedpositive for caspase 3 & 7 when the cells were incubated in medium 1,seen as asterisk in FIG. 13(A), compared to a decreased number ofcaspase 3&7 positive cells when grown in medium 2, seen as asterisk inFIG. 13(B).

Cell viability was then tested against other blood serum. hUCB cellswere collected as discussed in Example 1. The cells were cultured inmedia supplemented with cord blood plasma, adult human serum (humanserum), or fetal bovine serum (FBS). Cells were incubated for 3 days,followed by a PBS wash and analysis of viability using the commerciallyavailable LIVE/DEAD cell viability assay (ThermoFisher Scientific, Cat.No. L3224). Six random fields were selected and images by confocalmicroscopy for each growth condition. As seen in FIG. 14 , cells grownin human serum possessed the lowest number of live cells, with around 62live cells identified. By comparison, cells grown in cord blood plasmaand fetal bovine serum were found to have around 85 live cells and 88live cells, respectively. The number of dead cells was found to be thehighest in fetal bovine serum-supplemented media, followed by humanserum and cord blood, at 79 cells, around 25 cells, and around 22 cells,respectively. This resulted in a ratio of live to dead cells of 3.7:1for cord blood plasma, 2.4:1 for human serum, and 1.1:1 for fetal bovineserum. Cell viability was calculated, following a similar pattern, withcord blood showing viability of around 79%, 70% for human serum, andaround 52% for fetal bovine serum, as seen in FIG. 15 .

Cord blood plasma decreased apoptotic Caspase 3&7 activity in MNCsisolated from the peripheral blood of patients with both abnormalextensive or non-inducible cell proliferation to the mitogen (PHA).

Example 6

Intravenous administration of hUCB cells delayed the progression ofdisease and prolonged lifespan in the G93A SOD1 mouse model of ALS(Garbuzova-Davis, et al., Multiple intravenous administrations of humanumbilical cord blood cells benefit in a mouse model of ALS. PLoS One7(2):e31254; 2012; Garbuzova-Davis, et al., Human umbilical cord bloodtreatment in a mouse model of ALS: optimization of cell dose. PLoS One3(6):e2494; 2008; Garbuzova-Davis, et al., Intravenous administration ofhuman umbilical cord blood cells in a mouse model of amyotrophic lateralsclerosis: distribution, migration, and differentiation. J. Hematother.Stem Cell Res. 12(3):255-270; 2003). These results were furthersupported by observations of increased motor neuron survival in both thecervical and lumbar regions of the spinal cord. Also, restored WBCprofiles and decreased pro-inflammatory cytokine production weredetermined. While these results have yet to be replicated in the clinic,the results demonstrate the therapeutic potential of using plasmaderived from hUCB to mitigate the mitogen-induced proliferation responseof MNCs isolated from the peripheral blood of ALS patients in vitro.

ALS patients differed in lymphocyte functionality, possibly due todifferences in the immune response as a consequence of the diseasestate. The patient with an abnormally extensive cell proliferation inresponse to mitogen (PHA) in vitro (Group III) may result from anautoimmunity impairment while the non-inducible proliferation patients(Group II) suggests immune deficiency.

This suggests that use of therapies which affect the immune system maynot be effective in all patients, suggesting that a more personalizedmedicine approach may be necessary. A recent clinical study ofautologous MSCs as a treatment therapy for ALS suggested that not allpatients responded to treatment (Kim, et al., Biological markers ofmesenchymal stromal cells as predictors of response to autologous stemcell transplantation in patients with amyotrophic lateral sclerosis: aninvestigator-initiated trial and in vivo study. Stem Cells32(10):2724-2731; 2014). A higher secretion of biological markers suchas VEGF, angiopoietin and TGF-β was observed from the MSCs of thosepatients who responded to the treatment and this could be exploredfurther with regards to the observations.

Innate and adaptive immune responses clearly play an important role inALS. Infiltration of microglia and T cells is evident, and it has beensuggested that these cells may initially be protective (Banerjee, etal., Adaptive immune neuroprotection in G93A-SOD1 amyotrophic lateralsclerosis mice. PLoS One3(7):e2740; 2008; Beers, et al., CD4+ T cellssupport glial neuroprotection, slow disease progression, and modifyglial morphology in an animal model of inherited ALS. Proc. Natl. Acad.Sci. USA 105(40):15558-15563; 2008; Chiu, et al., T lymphocytespotentiate endogenous neuroprotective inflammation in a mouse model ofALS. Proc. Natl. Acad. Sci. USA 105(46):17913-17918; 2008), but somestudies have also observed lymphopenia in ALS patients or G93A SOD1symptomatic mice (Banerjee, et al., Adaptive immune neuroprotection inG93A-SOD1 amyotrophic lateral sclerosis mice. PLoS One3(7):e2740; 2008;Kuzmenok, et al., Lymphopenia and spontaneous autorosette formation inSOD1 mouse model of ALS. J. Neuroimmunol. 172(1-2):132-136; 2006;Provinciali, et al., Immunity assessment in the early stages ofamyotrophic lateral sclerosis: a study of virus antibodies andlymphocyte subsets. Acta Neurol. Scand. 78(6):449-454; 1988). However,the precise roles of the immune responses, whether causative and/or aconsequence of the disease still need to be determined (Murdock, et al.,The dual roles of immunity in ALS: injury overrides protection.Neurobiol. Dis. 77:1-12; 2015; Rodrigues, et al., The innate andadaptive immunological aspects in neurodegenerative diseases. J.Neuroimmunol. 269(1-2):1-8; 2014). While there is no doubt that theimmune system is involved in ALS, it is worth noting thatimmunosuppressive therapies for ALS are not very effective (Pagani, etal., Autoimmunity in amyotrophic lateral sclerosis: past and present.Neurol. Res. Int. 2011:497080; 2011). There is evidence for autoimmunitybeing a component of ALS, though it is unclear whether it is causativeor an epiphenomenon (Alexianu, The role of immune processes inamyotrophic lateral sclerosis pathogenesis. Rom. J. Neurol. Psychiatry33(3-4):215-227; 1995; Appel, et al., Autoimmunity as an etiologicalfactor in sporadic amyotrophic lateral sclerosis. Adv. Neurol. 68:47-57;1995; Coban, et al., Serum anti-neuronal antibodies in amyotrophiclateral sclerosis. Int. J. Neurosci. 123(8):557-562; 2013;Niebroj-Dobosz, et al., Auto-antibodies against proteins of spinal cordcells in cerebrospinal fluid of patients with amyotrophic lateralsclerosis (ALS). Folia Neuropathol. 44(3):191-196; 2006; Pagani, et al.,Autoimmunity in amyotrophic lateral sclerosis: past and present. Neurol.Res. Int. 2011:497080; 2011; Rodrigues, et al., The innate and adaptiveimmunological aspects in neurodegenerative diseases. J. Neuroimmunol.269(1-2):1-8; 2014), with some suggestion that autoimmunity could bebeneficial in chronic neuroinflammation (Schwartz & Baruch, Breakingperipheral immune tolerance to CNS antigens in neurodegenerativediseases: boosting autoimmunity to fight-off chronic neuroinflammation.J. Autoimmun. 54:8-14; 2014). Serum, CSF and immune cells from ALSpatients has also been shown to contain increased levels of IL-17 andIL-23, which may be a sign of T helper 17 (Th17) cell activation—a celltype that may play a crucial role in destructive autoimmunity (Fiala, etal., IL-17A is increased in the serum and in spinal cord CD8 and mastcells of ALS patients. J. Neuroinflammation 7:76; 2010; Rentzos, et al.,Interleukin-17 and interleukin-23 are elevated in serum andcerebrospinal fluid of patients with ALS: a reflection of Th17 cellsactivation? Acta Neurol Scand. 122(6):425-429; 2010; Saresella, et al.,T helper-17 activation dominates the immunologic milieu of bothamyotrophic lateral sclerosis and progressive multiple sclerosis. Clin.Immunol. 148(1):79-88; 2013).

While the study demonstrated impairment of mononuclear cells obtainedfrom the peripheral blood of ALS patients via mitogen induction,Bossolasco et al. (Bossolasco, et al., Metalloproteinase alterations inthe bone marrow of ALS patients. J. Mol. Med. 88(6):553-564; 2010) havedetected impaired functionality of bone marrow stem cells (BMSCs) fromALS patients in the ability to proliferate and differentiate intoadipogenic and osteoblastic tissue, though Ferrero et al. (Ferrero, etal., Bone marrow mesenchymal stem cells from healthy donors and sporadicamyotrophic lateral sclerosis patients. Cell Transplant. 17(3):255-266;2008) noted no significant differences in the proliferation potential ofbone marrow mesenchymal stem cells from ALS patients. Liu and Martin(Liu, & Martin, The adult neural stem and progenitor cell niche isaltered in amyotrophic lateral sclerosis mouse brain. J. Comp. Neurol.497(3):468-88; 2006) showed a similar impairment of neural stem cells(NSCs) in the subventricular zone of symptomatic G93A SOD1 mice. Thesestudies suggested that some cell populations, such as the peripheralblood lymphocytes and possibly the BMSCs, undergo changes in theirability to proliferate and/or differentiate in ALS patients, however, noreports exist to confirm any abnormal cell function. Though Kang et al.(Kang, et al., Degeneration and impaired regeneration of gray matteroligodendrocytes in amyotrophic lateral sclerosis. Nat. Neurosci.16(5):571-579; 2013) have detected enhanced proliferation ofnon-stimulated oligodendrocytic progenitors in the G93A SOD1 transgenicmouse.

The findings demonstrated that cord blood plasma was effective atmodulating the cell response to PHA in the patient with abnormallyextensive cell proliferation (Group III) as well as the patients withnon-inducible cell proliferation (Group II), but not the patients whoresponded normally (Group I). Also, hUCBP decreased apoptotic Caspase3/7 activity in MNCs isolated from the peripheral blood of patients withboth abnormal extensive or non-inducible cell proliferation to themitogen (PHA). Additionally, when standard media (Medium 1) was replacedwith media containing hUCBP (Medium 2) the apoptotic activity of theMNCs in culture tended to decrease. These findings reinforce the currentanti-inflammatory observations that have been made of hUCB cells(Garbuzova-Davis, et al., Multiple intravenous administrations of humanumbilical cord blood cells benefit in a mouse model of ALS. PLoS One7(2):e31254; 2012), and also demonstrate that plasma derived from cordblood could be an effective treatment in ALS patients with immunedysfunction as an immune-modulator and/or anti-apoptotic factor.

ALSFRS/ALSFRS-R scoring of ALS patients is a well-recognized and widelyused standard in ALS clinics to validate patient disease stage. Althoughthe testing methodology might be subjective, all the data was collectedby the same neurologist in order to minimize the potential for bias. Thescores were then calculated using the on-line ALS C.A.R.E. program(Center for Outcomes Research, Univ Massachusetts Medical School, 2015).

Although the patient sample size in the study was modest, it wassufficient to provide a valid analysis of hUCB plasma effects onmitogen-induced proliferation of MNCs isolated from the peripheral bloodof ALS patients. Additionally, the significant reduction of apoptoticactivity of these cells via hUCB plasma is an important study finding.

The therapeutic uses of hUCB plasma (hUCBP) are shown for ALS. hUBCPmodulates immune cell response to stimulation with the mitogen PHA.Also, hUCBP is a novel therapy that appears to correct any immunologicalissues that arise from ALS. This therapy can be combined with hUCB cell(or other cell) transplants to potentially help provide a moresupportive environment for the transplanted cells.

Example 7

In development of alternative approaches in treatment for age-relateddiseases, proteins from “young” blood have been intensely investigated.Studies of parabiosis, with shared blood circulatory systems between old(16-20 months of age) and young (2-3 months of age) mice, have shownsignificantly improved cognition and physical function in both agedwild-type mice14 and a mouse model of Alzheimer's disease (AD). (VilledaS A, Plambeck K E, Middeldorp J, et al. Young blood reverses age-relatedimpairments in cognitive function and synaptic plasticity in mice. NatMed. 2014; 20:659-663; Middeldorp J, Lehallier B, Villeda S A, et al.Preclinical assessment of young blood plasma for Alzheimer disease. JAMANeurol. 2016; 73:1325-1333). Middeldorp et al demonstrated thatparabiosis of young wild-type mice with AD mice for 5 weeks effectivelyimproved learning and memory while also reducing inflammation in ADmice. (Middeldorp J, Lehallier B, Villeda S A, et al. Preclinicalassessment of young blood plasma for Alzheimer disease. JAMA Neurol.2016; 73:1325-1333). Additionally, the authors noted increased synapticactivity in the hippocampus of AD mice. Based on these study results,clinical trial (NCT02256306) investigated the safety of 4-weeklyinfusions of young blood plasma from donors aged between 18 and 30 yearsof age into patients with AD. Although no serious adverse reactionsoccurred, the study found no significant effect on patient cognition butdid show significant improvements in daily living skills.

Although results of using young blood are promising, it is still unclearwhich constituents of “young” blood are providing beneficial effects.Potentially, paracrine actions are involved in positive outcomes fortreatment of an age-related disease such as AD. Also, hormonal status ofdonors should be investigated due to the wide age range (18-30 years) ofdonors. Alternatively, plasma derived from hUCB could be a morebeneficial therapeutic due to its unique and uniform molecularcomposition.

In the present study, various factors in CBP derived from hUCB and theeffect of CBP on mononuclear cells isolated from hUCB (MNC hUCB) invitro were evaluated in the context of establishing CBP as a potentialtherapeutic agent. Cytokine and growth factor profiles were examinedwithin the same samples of CBP and human adult blood plasma/sera(ABP/S). The effect of autologous CBP on MNC hUCB in vitro wasdetermined and compared to ABP/S and standard FBS media supplements. Themajor study findings were that CBP demonstrated: (a) significantly “low”concentrations of the proinflammatory cytokines IL-2, IL-6, IFN-γ, andTNF-α; (b) significantly “low” concentrations of immunomodulatory IL-5cytokine and GM-CSF; (c) significantly “elevated” level of the chemokineIL-8; (d) significantly high concentrations of VEGF, G-CSF, EGF andFGF-basic growth factors; (e) significantly “increased” viability of MNChUCB in vitro with autologous CBP media supplement; and (f)significantly “decreased” apoptotic MNC hUCB in vitro with autologousCBP media supplement.

The inventors are the first to demonstrate the unique CBP composition ofcytokines and growth factors within the same samples, providing evidenceof the unique protein content in CBP. Also, the inventors found thatautologous CBP promoted MNC hUCB viability and reduced apoptotic celldeath in vitro, supporting the notion that CBP has potential as a soletherapeutic or cell-additive agent in developing clinically relevantCBP-based therapies for various neurodegenerative diseases.

It has been shown that in addition to a high concentration of growthfactors, human CBP also contains a great amount of soluble proliferativeand immunomodulatory factors such as (TGF)-β, G-CSF, GM-CSF, monocytechemoattractant protein (MCP)-1, IL-6, and IL-8.17 Also, IL-16 cytokine,a modulator of T cell activation, has been detected in CBP18 andpotentially presents a physiological mechanism for fetal-maternaltolerance. (Tekkatte C, Gunasingh G P, Cherian K M, Sankaranarayanan K.“Humanized” stem cell culture techniques: the animal serum controversy.Stem Cells Int. 2011). Due to CBP's specific molecular composition,numerous studies showed beneficial effect of CBP in replacement ofstandard FBS for various cell expansions in vitro, which may beessential to achieve appropriate cell numbers for clinical use. (Ding Y,Yang H, Feng J B, et al. Human umbilical cord-derived MSC culture: thereplacement of animal sera with human cord blood plasma. In Vitro CellDev Biol Anim. 2013; 49:771-777; Lee J-Y, Nam H, Park Y-J, et al. Theeffects of platelet-rich plasma derived from human umbilical cord bloodon the osteogenic differentiation of human dental stem cells. In VitroCell Dev Biol Anim. 2011; 47:157-164; Kim Y-M, Jung M-H, Song H-Y, etal. Ex vivo expansion of human umbilical cord blood-derivedT-lymphocytes with homologous cord blood plasma. Tohoku J Exp Med. 2005;205:115-122; Huang L, Critser P J, Grimes B R, Yoder M C. Humanumbilical cord blood plasma can replace fetal bovine serum for in vitroexpansion of functional human endothelial colony-forming cells.Cytotherapy. 2011; 13:712-721).

In the current study, cytokine and growth factor levels were analyzed inthe same CBP samples for a better understanding of CBP molecularcomposition prior to proposing CBP as a therapeutic agent. The inventorsshowed low concentrations of pro-inflammatory IL-2, IL-6, IFN-γ andTNF-α cytokines in CBP, presumably secreted by various cells in hUCB,which signify the immune immaturity of these cell populations.

Additionally, the study findings demonstrated a significantly lowconcentration of immunomodulatory cytokine IL-5 in CBP vs. ABP/S,supporting previous study results. (Garanina E E, Gatina D, Martynova EV, et al. Cytokine profiling of human umbilical cord plasma and humanumbilical cord blood mononuclear cells. Blood. 2017; 130:4814). Thiscytokine, mainly produced by Th2 helper lymphocytes and mast cells,promotes growth/differentiation of B cells and granulocytes uponimmunological and/or antigenic priming in development of the adaptiveimmune response. However, increased concentrations of IL-5, IL-2 andtranscription factor GATA-4 determined in CBP may result in abnormalpatterns of fetal immune system development and induce risk of allergicdisease. (Marschan E, Honkanen J, Kukkonen K, et al. Increasedactivation of GATA-3, IL-2 and IL-5 of cord blood mononuclear cells ininfants with IgE sensitization. Pediatr Allergy Immunol. 2008;19:132-139).

Also, it has been shown that antioxidant capacity, evaluated by carbonyllevels in CBP, was significantly higher in patients delivering neonatesby caesarean vs. vaginal route, suggesting that the delivery methodimpacts oxidative stress. (Noh E J, Kim Y H, Cho M K, et al. Comparisonof oxidative stress markers in umbilical cord blood after vaginal andcesarean delivery. Obstet Gynecol Sci. 2014; 57:109-114). In the presentstudy, the low concentration of GM-CSF found in CBP together with thelow concentrations of pro-inflammatory cytokines provide furtherevidence of anti-inflammatory hUCB content. Thus, low levels ofpro-inflammatory and immunomodulatory cytokines in CBP provide afavorable microenvironment for cellular content in hUCB. It has beenshown that transplantation of MNC derived from hUCB even from unrelateddonors into patients with hematologic malignancies causes a lowincidence of graft-versus-host disease compared to bone marrow orperipheral blood cell administration. (Zhang H, Chen J, Que W. Ameta-analysis of unrelated donor umbilical cord blood transplantationversus unrelated donor bone marrow transplantation in acute leukemiapatients. Biol Blood Marrow Transplant. 2012; 18:1164-1173; Chen Y, XuL, Liu D, et al. Comparative outcomes between cord blood transplantationand bone marrow or peripheral blood stem cell transplantation fromunrelated donors in patients with hematologic malignancies: asingle-institute analysis. Chin Med J. 2013; 126:2499-2503).

The present study results also demonstrated similar amounts ofanti-inflammatory IL-4 and IL-10 cytokines in CBP and ABP/S, However, itis important to note that these anti-inflammatory cytokines were presentat a greater concentration than the pro-inflammatory constituents ofCBP, suggesting a favorable cytokine composition towards developing CBPas potential therapeutic agent. Since IL-10 is an important cytokine fordownregulation of Th1 inflammatory cytokines and MHC class II antigens,a decrease of this cytokine is mainly associated with alteredcell-mediated immunosuppression and induction of complications duringpregnancy. (Mobini M, Mortazavi M, Nadi S, et al. Significant rolesplayed by interleukin-10 in outcome of pregnancy. Iran J Basic Med Sci.2016; 19:119-124). In contrast, increased cord blood IL-10 wasdetermined in preterm infants compared to full-term newborns.(Blanco-Quirós A, Arranz E, Solis G, et al. Cord blood interleukin-10levels are increased in preterm newborns. Eur J Pediatr. 2000;159:420-423; Blanco-Quirós A, Arranz E, Solis G, et al. High cord bloodIL-10 levels in preterm newborns with respiratory distress syndrome.Allergol Immunopathol (Madr). 2004; 32:189-196). In the current study,hUCB units were used from healthy infants delivered naturally, so IL-10levels determined in CBP vs. ABP/S likely reflect steadyimmune/inflammatory humoral status in hUCB.

Amongst the additional important study findings were significantelevations of VEGF, G-CSF, EGF and FGF-basic growth factors in CBP vs.ABP/S. Both EGF and FGF-basic promote stem cell renewal and inhibit cellsenescence and elevated levels of EGF largely correlate to gestationalage and birth weight of the developing fetus. (Coutu D L, Galipeau J.Roles of FGF signaling in stem cell self-renewal, senescence and aging.Aging (Albany N.Y.). 2011; 3:920-933; Ichiba H, Fujimura M, Takeuchi T.Levels of epidermal growth factor in human cord blood. Biol Neonate.1992; 61:302-307; Wahab Mohamed W A, Aseeri A M. Cord blood epidermalgrowth factor as a possible predictor of necrotizing enterocolitis invery low birth weight infants. J Neonatal Perinatal Med. 2013;6:257-262). Thus, the increased levels of EGF and FGF-basic in CBPdetermined in the study may indicate normal fetal development. Also,increased G-CSF, a bone marrow stem cell mobilizing factor, in CBPpotentially reflects intensive production of bone marrow derived stemcells in the fetus. The combination of this growth factor with MNC hUCBfor the treatment of myeloid malignancies in human adults afterradiation promoted cell engraftment in bone marrow replacementtherapies. (Delaney C, Ratajczak M Z, Laughlin M J. Strategies toenhance umbilical cord blood stem cell engraftment in adult patients.Expert Rev Hematol. 2010; 3:273-283; Broxmeyer H E, Hangoc G, Cooper S,et al. Growth characteristics and expansion of human umbilical cordblood and estimation of its potential for transplantation in adults.Proc Natl Acad Sci USA. 1992; 89:4109-4113). Also, co-administration ofG-CSF with MNC hUCB into an animal model of traumatic brain injuryresults demonstrated reduction of neuroinflammation and promotion ofstem cells into the injured side of the brain. (De La Peña I, Sanberg PR, Acosta S, et al. G-CSF as an adjunctive therapy with umbilical cordblood cell transplantation for traumatic brain injury. Cell Transplant.2015; 24:447-457).

Of note, significantly elevated levels of the chemokine IL-8 and VEGFwere determined in CBP vs. ABP/S in the current study. While IL-8 isprimarily known as a pro-inflammatory mediator, it also recognized as apromoter of angiogenic activity as demonstrated by endothelial cellsurvival, proliferation and migration in vitro. (Li A, Dubey S, Varney ML, et al. IL-8 directly enhanced endothelial cell survival,proliferation, and matrix metalloproteinases production and regulatedangiogenesis. J Immunol. 2003; 170:3369-3376; Lai Y, Liu X H, Zeng Y, etal. Interleukin-8 induces the endothelial cell migration through the Rac1/RhoA-p38MAPK pathway. Eur Rev Med Pharmacol Sci. 2012; 16:630-638).Interestingly, the concentration of the angiogenic VEGF growth factorwas also significantly higher in CBP vs. ABP/S. It is possible that theelevated level of VEGF is a result of the high concentration of IL-8,which promotes increased expression of VEGF by endothelial cells.(Martin D, Galisteo R, Gutkind J S. CXCL8/IL8 stimulates vascularendothelial growth factor (VEGF) expression and the autocrine activationof VEGFR2 in endothelial cells by activating NFkappaB through the CBM(Carma3/Bcl10/Malt1) complex. J Biol Chem. 2009; 284: 6038-6042; Li M,Zhang Y, Feurino L W, et al. Interleukin-8 increases vascularendothelial growth factor and neuropilin expression and stimulates ERKactivation in human pancreatic cancer. Cancer Sci. 2008; 99:733-737). Arecently published study demonstrated that microRNA-containing exosomesderived from maternal and umbilical cord serum dramatically promotehuman umbilical vein endothelial cell proliferation, migration, and tubeformation in vitro, highlighting the important role of exosomes in theregulation of angiogenesis during gestation. (Jia L, Zhou X, Huang X, etal. Maternal and umbilical cord serum derived exosomes enhanceendothelial cell proliferation and migration. FASEB J. 2018).Exclusively, VEGF has been studied for potential therapeutic efficacy inanimal models of ALS and its use in clinical settings has been discussed(Lambrechts D, Storkebaum E, Carmeliet P. VEGF: necessary to preventmotoneuron degeneration, sufficient to treat ALS?Trends Mol Med. 2004;10:275-282; Pronto-Laborinho A C, Pinto S, de Carvalho M. Roles ofvascular endothelial growth factor in amyotrophic lateral sclerosis.Biomed Res Int. 2014; Keifer O P, O'Connor D M, Boulis N M. Gene andprotein therapies utilizing VEGF for ALS. Pharmacol Ther. 2014;141:261-271). Nevertheless, CBP containing high levels of IL-8 and VEGFmight be a beneficial treatment for repair of the damaged blood-brainbarrier and/or blood-spinal cord barrier in patients with ALS, AD,Parkinson's disease and multiple sclerosis. (Garbuzova-Davis S,Hernandez-Ontiveros D G, Rodrigues M C O, et al. Impairedblood-brain/spinal cord barrier in ALS patients. Brain Res. 2012;1469:114-128; Garbuzova-Davis S, Sanberg P R. Blood-CNS barrierimpairment in ALS patients versus an animal model. Front Cell Neurosci.2014; 8:21; Henkel J S, Beers D R, Wen S, et al. Decreased mRNAexpression of tight junction proteins in lumbar spinal cords of patientswith ALS. Neurology. 2009; 72:1614-1616; Winkler E A, Sengillo J D,Sullivan J S, et al. Blood-spinal cord barrier breakdown and pericytereductions in amyotrophic lateral sclerosis. Acta Neuropathol. 2013;125:111-120; Goos J D C, Teunissen C E, Veerhuis R, et al. Microbleedsrelate to altered amyloid-β metabolism in Alzheimer's disease. NeurobiolAging. 2012; Kortekaas R, Leenders K L, van Oostrom J C H, et al.Blood-brain barrier dysfunction in parkinsonian midbrain in vivo. AnnNeurol. 2005; 57:176-179; Stone L A, Smith M E, Albert P S, et al.Blood-brain barrier disruption on contrast-enhanced MRI in patients withmild relapsing-remitting multiple sclerosis: relationship to course,gender, and age. Neurology. 1995; 45:1122-1126).

Finally, the in vitro studies showed significantly increased viabilityof MNC hUCB when autologous CBP was added to culture media. Also,apoptotic activity of MNC hUCB in vitro, determined by TUNEL, was alsodecreased after autologous CBP exposure compared to culturessupplemented with ABP/S or FBS. Supporting this novel finding, theprevious study demonstrated reduced activities of other pro-apoptoticfactors, such as caspase 3/7, from ALS patient-derived MNC's cultured inmedia supplemented with CBP. (Eve D J, Ehrhart J, Zesiewicz T, et al.Plasma Derived From Human Umbilical Cord Blood Modulates Mitogen-InducedProliferation of Mononuclear Cells Isolated From the Peripheral Blood ofALS Patients. Cell Transplant. 2016; 25:963-971). In this context,numerous studies have shown neuroprotective effects of MNC hUCBadministered into animal models of ALS, AD, Parkinson's disease,ischemic stroke and traumatic brain injury. (Ende N, Weinstein F, ChenR, Ende M. Human umbilical cord blood effect on sod mice (amyotrophiclateral sclerosis). Life Sci. 2000; 67:53-59; Garbuzova-Davis S, WillingA E, Zigova T, et al. Intravenous administration of human umbilical cordblood cells in a mouse model of amyotrophic lateral sclerosis:distribution, migration, and differentiation. J Hematother Stem CellRes. 2003; 12:255-270; Garbuzova-Davis S, Sanberg C D, Kuzmin-Nichols N,et al. Human umbilical cord blood treatment in a mouse model of ALS:optimization of cell dose. PLoS ONE. 2008; Garbuzova-Davis S, RodriguesM C O, Mirtyl S, et al. Multiple intravenous administrations of humanumbilical cord blood cells benefit in a mouse model of ALS. PLoS ONE.2012; Nikolic W V, Hou H, Town T, et al. Peripherally administered humanumbilical cord blood cells reduce parenchymal and vascular beta amyloiddeposits in Alzheimer mice. Stem Cells Dev. 2008; 17:423-439; DarlingtonD, Deng J, Giunta B, et al. Multiple low-dose infusions of humanumbilical cord blood cells improve cognitive impairments and reduceamyloid-β-associated neuropathology in Alzheimer mice. Stem Cells Dev.2013; 22:412-421; Darlington D, Li S, Hou H, et al. Human umbilical cordblood-derived monocytes improve cognitive deficits and reduce amyloid-βpathology in PSAPP mice. Cell Transplant. 2015; 24:2237-2250; Abo-GrishaN, Essawy S, Abo-Elmatty D M, Abdel-Hady Z. Effects of intravenous humanumbilical cord blood CD34+ stem cell therapy versus levodopa inexperimentally induced Parkinsonism in mice. Arch Med Sci. 2013;9:1138-1151; Newcomb J D, Ajmo C T, Sanberg C D, et al. Timing of cordblood treatment after experimental stroke determines therapeuticefficacy. Cell Transplant. 2006; 15:213-223; Boltze J, Schmidt U R,Reich D M, et al. Determination of the therapeutic time window for humanumbilical cord blood mononuclear cell transplantation followingexperimental stroke in rats. Cell Transplant. 2012; 21:1199-1211; AcostaS A, Tajiri N, Shinozuka K, et al. Combination therapy of humanumbilical cord blood cells and granulocyte colony stimulating factorreduces histopathological and motor impairments in an experimental modelof chronic traumatic brain injury. PLoS ONE. 2014; Min K, Song J, Lee JH, et al. Allogenic umbilical cord blood therapy combined witherythropoietin for patients with severe traumatic brain injury: threecase reports. Restor Neurol Neurosci. 2013; 31:397-410). However,insignificant numbers of MNC hUCB were detected in the CNS of theseanimal models after intravenous cell administration. This scarcity islikely due to a low rate of cell survival, since cell preparation andinjection involve dilution with a basic buffer solution. Substitution ofthis diluent with autologous CBP presents a more supportivemicroenvironment for cell survival and increases therapeutic efficacy ofadministered MNC hUCB. Especially, complementing MNC hUCB withautologous CBP may foster injected cell survival as supported by the invitro study results on cell viability and apoptotic activity. Also,repeated administrations of MNC hUCB cells with autologous CBP may proveeven more advantageous. Alternatively, injection of non-autologous CBPalone can be efficacious for treatment of various neurodegenerativediseases and/or aging population per se. Beneficial effects have beenobserved from intravenous administration of CBP into rats modellingacute ischemic stroke or into an animal model of aging. (Yoo J, Kim H-S,Seo J-J, et al. Therapeutic effects of umbilical cord blood plasma in arat model of acute ischemic stroke. Oncotarget. 2016; 7:79131-79140;Castellano J M, Mosher K I, Abbey R J, et al. Human umbilical cordplasma proteins revitalize hippocampal function in aged mice. Nature.2017; 544:488-492). In these studies, multiple injections of CBP wereperformed and this therapeutic approach needs to be considered. Inagreement with this approach, repeated deliveries of CBP could provideongoing trophic support for damaged cells and/or tissues. The inventorsshowed that CBP is a potential therapeutic due to its uniquecomposition. The inventors examine the effect of CBP alone and incombination with MNC hUCB for treatment of ALS using a symptomaticanimal model of disease for a translational perspective.

Results

Cord Blood Plasma Cytokine Profile

Samples of CBP and ABP/S were assayed to determine cytokine profilesusing an ultrasensitive human cytokine 10-plex panel. Results showedsignificantly (P<0.01) lower concentrations of the proinflammatorycytokines IL-2, IFN-γ and TNF-α in CBP compared to ABP/S (FIG. 16B,H,I).Additionally, levels of immunomodulatory IL-5 (FIG. 16D) andmultifunctional IL-6 (FIG. 16E) cytokines were also significantly(P<0.01) lower in CBP vs. ABP/S. Significantly (P<0.01) elevatedconcentrations of the chemokine IL-8 were determined in CBP incomparison in ABP/S (FIG. 16F). Interestingly, levels of thepro-inflammatory immune cell maturating factor, GMCSF, weresignificantly (P<0.01) lower in CBP than in ABP/S (FIG. 16J). Althoughthe levels of IL-1β, IL-4 and IL-10 were slightly reduced in CBPcompared to ABP/S, these reductions were not statistically significant(P>0.05) (FIG. 16A,C,G). While anti-inflammatory IL-4 and IL-10 cytokineconcentrations were not significantly different between CBP and ABP/S,it is important to note that most of the pro-inflammatory cytokineswithin CBP were present at lower concentrations than theiranti-inflammatory counterparts. Concentrations of cytokines in CBP andABP/S are provided in Table 1A of FIG. 17 .

Cord Blood Plasma Growth Factor Profile

The levels of several common growth factors were measured in CBP andABP/S using a human growth factor four-plex assay. The concentrations ofVEGF were significantly (P<0.01) higher in CBP, more than two-fold, vs.ABP/S (FIG. 18A). The concentrations of G-CSF, a bone marrow stem cellstimulating growth factor, were also significantly (P<0.05) higher inCBP compared to ABP/S (FIG. 18B). Also, the cell proliferating growthfactors: epidermal growth factor (EGF) and fibroblast growth factorbasic (FGF-basic) were significantly (P<0.01) elevated in CBP (FIG.18C,D; respectively). Of note, the levels of EGF and FGF-basic factorswere about 2.5-fold higher in CBP vs. ABP/S. Levels of growth factors inCBP and ABP/S are indicated in Table 1B of FIG. 17 .

Viability of MNC hUCB Cultured with Autologous CBP

Cryopreserved MNC hUCB was incubated with RPMI-1640 media supplementedwith autologous CBP, ABP/S, or FBS for 5 days. After 5 days in vitro,the cells were stained using the LIVE/DEAD Viability/Cytotoxicity assayto identify the viable (dark grey) and non-viable cytotoxic cellpopulations (light grey). Numerous viable MNC hUCB were observed incultures with CBP (FIG. 19Aa) and FBS (FIG. 19Ac) supplements. Fewerviable cells were seen with ABP/S (FIG. 19Ab) added into media. Livecell counts of MNC hUCB supplemented with autologous CBP weresignificantly (83.83±10.86 cell number, P<0.05) higher vs. culturedcells supplemented with ABP/S (60.35±5.50 cell number, FIG. 19B).However, numbers of viable cells cultured with CBP (83.83±10.86 cellnumber) and FBS (87.33±7.17 cell number) were similar (FIG. 19B).Importantly, media supplemented with CBP showed significantly (P<0.01)reduced numbers of dead MNC hUCB (22.50±3.67 cell number) compared toFBS (79.33±10.48 cell number). Yet, MNC hUCB cultured with FBSdemonstrated a significant (P<0.05) increase of dead cells vs. culturedcells supplemented with ABP/S (38.15±6.90 cell number, FIG. 19B).Additionally, cells supplemented in media with CBP had a greater ratioof live to dead cells (3.7:1) compared to cultures that received ABP/S(1.6:1) or FBS (1.1:1).

Apoptotic Activity of MNC hUCB Cultured with Autologous CBP

Apoptotic activity of cultured MNC hUCB in media supplemented withautologous CBP, ABP/S, or FBS was analyzed on day 5 in vitro using acolormetric TUNEL assay. The percentage of apoptotic cells cultured withCBP was significantly lower (17.39±1.70%) compared to culturessupplemented with ABP/S (34.72±2.61%, P<0.001) or FBS (26.62±2.08%,P<0.01) (FIG. 20A). Interestingly, MNC hUCB cultured in media containingFBS showed significantly (P<0.05) lower apoptotic activity vs. culturedcells with ABP/S. Phase contrast microscopic images of MNC hUCB in vitrodemonstrated a few cells with abnormal morphology displaying dislocatednuclei in cultures supplemented with CBP (FIG. 20Ba) compared tonumerous morphologically damaged cells cultured with ABP/S (FIG. 20Bb)or FBS (FIG. 20Bc), supporting apoptotic cell counts.

Materials and Methods

The human umbilical cord blood (hUCB) units were collected by Texas CordBlood Bank (TCBB, GenCure, West San Antonio, Tex., USA) and provided toSaneron CCEL Therapeutics, Inc. for research purposes. The cord bloodunits were obtained from full-term pregnancies by vaginal delivery. Theumbilical cord blood units were received within 48 hours of collection.Maternal blood samples, collected as the same time as the cord blood,were tested by TCBB for infectious disease markers of HIV, hepatitis Band C, syphilis, CMV and HTLV I&II, and test results were provided forvalidation of the cord blood units. Each cord blood unit in the studywas negative for all infectious disease markers as determined inmaternal blood. Human adult blood plasma or sera (ABP/S) was obtainedfrom a commercially available source (Sigma-Aldrich, St. Louis, Mo.,USA). Upon receipt of ABP/S, samples were aliquoted and stored at −20°C.

Human Umbilical Cord Blood Processing and Plasma Isolation

Human umbilical cord blood (hUCB) units (n=20), with maternal bloodsamples negative for all tested infectious markers, were processed toobtain an autologous CBP fraction and mononuclear cell population (MNChUCB, U-CORD-CELL™, Saneron CCEL Therapeutics, Inc., Tampa, Fla., USA)as detailed below. Upon receipt, the cord blood units were diluted (1:1)with sterile phosphate buffered saline (PBS) without Mg2+ orCa2+(Sigma-Aldrich, St. Louis, Mo., USA). The cord blood was thenfractionated using the density gradient solution Ficoll (Ficoll-PaquePremium: 1.078 g/mL, Cat. No. 17544202; Millipore Sigma, St. Louis, Mo.,USA) in the Sepax 2 fully automated cell processing system (BiosafeAmerica Inc., Houston, Tex., USA). This allowed for the sterilecollection of both CBP and MNC hUCB from each unit of cord blood. TheCBP was further centrifuged at 3000 g for 10 minutes to remove anyadditional red blood cells. The CBP was then aliquoted and stored at−20° C. The MNC hUCB cell numbers and viability were determined usingthe Vi-CELL Viability Analyzer (Beckman Coulter, Brea, Calif., USA). MNChUCB was then frozen at 5×10⁷ cells per vial using a proprietarycryopreservation media (Saneron CCEL Therapeutics, Inc.) and stored inliquid nitrogen.

Cytokine Profile in Human Umbilical Cord Blood Plasma

A human ultrasensitive cytokine 10-plex panel (Invitrogen, Carlsbad,Calif., USA; Cat. No. LHC6004) was used as previously described 13 todetermine the concentrations of cytokines within CBP (n=20) and ABP/S(n=6) in triplicate, following the manufacturer's protocol. Allmeasurements were performed by an investigator blinded to the samplesource. Granulocyte-macrophage colony-stimulating factor (GM-CSF) andcytokine levels of interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8,IL-10, interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) andGM-CSF were quantified using the Bio-Rad Bio-Plex® Luminex 200 multiplexassay system (Bio-Rad Laboratories Inc., Hercules, Calif., USA). TheBio-Rad Bio-Plex® 200 software (BioRad Laboratories Inc., HerculesCalif., USA) was used to calculate the sample cytokine concentrationsaccording to a standard curve and results were presented as picograms ofanalyte per milliliter (pg/mL).

Growth Factor Profile in Human Umbilical Cord Blood Plasma

A human growth factor 4-plex panel (Invitrogen; Cat No. LHC0007) wasemployed to determine various growth factor levels within CBP (n=20) andABP/S (n=6) samples in triplicate, following the manufacturer'sprotocol. All measurements were performed by an investigator blinded tothe source of the samples. Levels of VEGF, granulocytecolony-stimulating factor (G-CSF), epidermal growth factor (EGF) andfibroblast growth factor basic (FGF-basic) were determined using theBio-Rad Bio-Plex® Luminex 200 multiplex assay (BioRad Laboratories Inc.,Hercules Calif., USA). The Bio-Rad Bio-Plex® 200 software (BioRadLaboratories Inc., Hercules Calif., USA) was used to calculate thesample growth factor concentrations accordingly to a standard curve andresults were presented as pg/mL.

Viability of MNC hUCB Cultured with Autologous CBP

Cryopreserved MNC hUCB cells (n=4 units) were quickly thawed at 37° C.,washed with PBS, and centrifuged at 400 g for 5 minutes. Cell quantityand viability were determined using a hemocytometer. The cells were thenre-suspended with phenol-free RPMI-1640 media (Gibco, Dublin, Ireland;Cat. No. 11835030) and plated in a 24-well cell culture plate at adensity of 5×104 cells/well. Pre-designated wells were supplemented with10% of autologous CBP, ABP/S, or fetal bovine serum (FBS) (Gibco,Dublin, Ireland; Cat No. 10438026) upon initial plating in duplicate.Cells were incubated at 37° C. with 5% CO2 for 5 days. Media was changedat 24 hours and 3 days after cell plating. On day 5, cell viability wasdetermined using the LIVE/DEAD viability/cytotoxicity kit (MolecularProbes, Cat No. R37601) accordingly to the manufacturer's instructions.Briefly, the culture media was replaced with 250 μL of fresh PBS in eachwell. In an equal volume to PBS, LIVE/DEAD working solution (250 μL) wasadded to each well and incubated at 37° C. for 30 minutes.

After incubation, confocal microscopy images (n=3-4/well, totalingn=16-20/supplement, mainly from the middle of the well) of cellfluorescence were obtained at 10× magnification for cell quantificationusing the Olympus FluoView 1000 confocal laser scanning microscope(Olympus Corporation of the Americas, Center Valley, Pa., USA). Livecells were labelled with green fluorescence through the conversion ofnon-fluorescent cell-permanent calcein acetoxymethyl to intenselyfluorescent calcein by ubiquitous intracellular esterase enzymeactivity. Dead cells were identified using ethidium homodimer-1, whichenters cells through damaged membranes and produces a red fluorescenceupon binding to nucleic acids. Cell counts of live (green) and dead(red) cells were determined using NIH ImageJ software (version 1.46).

Apoptotic Activity of MNC hUCB Cultured with Autologous CBP

Cryopreserved MNC hUCB cells (n=6 units) were quickly thawed at 37° C.,washed with PBS, and centrifuged at 400 g for 5 minutes. Cell quantityand viability were determined using a hemocytometer. Cells were thenre-suspended with phenol-free RPMI-1640 media and plated in a 96-wellculture plate at a density of 2×104 cells/well. Pre-designated wellswere supplemented with 10% of either autologous CBP, ABP/S, or FBS uponinitial plating in duplicate. Cells were incubated at 37° C. with 5% CO2for 5 days. Media was changed at 24 hours and 3 days after cell plating.On day 5, the apoptotic activity of the cells was determined using theHT TiterTACS™ Assay kit (Trevigen, Bio-Techne, Minneapolis, Minn., USA;Cat No. 4822-96-K) accordingly to the manufacturer's instructions.Briefly, the cells were washed with 200 μL of sterile PBS, then quicklyfixed using a 3.7% PBS buffered formaldehyde solution. The cells werewashed once more with PBS and then permeabilized with Cytonin™ (50μL/well). TACS-Nuclease™ (50 μL/well) was then added to designated wellsto determine total absorbance. The plate was incubated for 60 minutes at37° C. following a wash with PBS. The endogenous peroxidase activity wasquenched with a 3% hydrogen peroxide solution. The wells were thenwashed once more with PBS and a 1×TdT labelling buffer reaction mix wasadded to the wells and incubated at 37° C. for 60 minutes. To stop thelabelling reaction, 1×TdT stop buffer was added to the well andincubated for 5 minutes, followed by a wash with PBS. Thestreptavidin-HRP enzyme solution was then added to the wells andincubated for 10 minutes at RT. After an additional wash with PBS, theTACS-Sapphire substrate solution was added and incubated for 30 minutesat RT with light protection. Stop solution of 0.2N HCl was added to eachwell and absorbance at 450 nm was measured using a spectrophotometer(SpectraMax Plus 384 microplate reader, Molecular Devices, LLC., SanJose, Calif., USA). Results were calculated as the percentage ofrelative apoptotic absorbance values to maximum absorbance valuesdetermined for each culture condition. Cell morphology was observedusing phase contrast images (n=6/supplement) obtained at 20× using anOlympus IX70 inverted microscope (Olympus Corporation of the Americas,Center Valley, Pa., USA).

Statistical Analysis

Data was presented as mean±SEM Statistical analysis was performed usingGraphPad Prism Software version 5 (GraphPad Software, Inc.). The resultsfor MNC hUCB viability and apoptotic activity were evaluated using aone-way ANOVA with Tukey's Multiple Comparison post-hoc test. Theresults for cytokine and growth factors in CBP were analyzed with atwo-tailed t test using same software. A value of P<0.05 was consideredsignificant.

CONCLUSION

In conclusion, the inventors demonstrate the unique protein content inthe same CBP samples composed of cytokines and growth factors. The novelin vitro finding of autologous CBP with MNC hUCB demonstrated thetrophic capacity of this combination through promotion of cell viabilityand reduction of apoptotic death. These findings further support thepotential of CBP as an independent therapeutic or cell-additive agent inclinical applications for various neurodegenerative diseases.

Example 8—Prophetic Treatment of ALS with Cord Blood Plasma

A human patient diagnosed with amyotrophic lateral sclerosis ispresented for treatment. The patient is treated with a therapeuticallyeffective amount of human umbilical cord blood plasma, prepared asdescribed below. The therapeutically effective amount of human umbilicalcord blood plasma is administered to the patient via intravenousinjection. Improvement is shown in motor behavior after treatment withthe cord blood plasma.

To increase improvement in motor behavior, the patient receivesadditional administrations of the same therapeutically effective amountof human umbilical cord blood plasma as multiple administrationsthroughout the treatment period. Improvement is shown in motor behavior.

Human umbilical cord blood units are obtained from full-term pregnanciesby vaginal delivery and are received within 48 hours of collection.Maternal blood samples, collected as the same time as the cord blood,are tested for infectious disease markers of HIV, hepatitis B and C,syphilis, CMV and HTLV I&II. Human umbilical cord blood (hUCB) units,with maternal blood samples negative for all tested infectious markers,are processed to obtain an autologous CBP fraction and mononuclear cellpopulation as detailed below. Upon receipt, the cord blood units arediluted (1:1) with sterile phosphate buffered saline (PBS) without Mg2+or Ca2+. The cord blood is then fractionated using the density gradientsolution Ficoll (Ficoll-Paque Premium: 1.078 g/mL, Cat. No. 17544202;Millipore Sigma, St. Louis, Mo., USA) in the Sepax 2 fully automatedcell processing system (Biosafe America Inc., Houston, Tex., USA), thusallowing for the sterile collection of both CBP and MNC hUCB from eachunit of cord blood. The CBP was further centrifuged at 3000 g for 10minutes to remove any additional red blood cells. The CBP was thenaliquoted and stored at −20° C. CBP is thawed prior to administration tothe patient.

Example 9—Prophetic Treatment of ALS with Cord Blood Plasma and hUCBCs

A human patient is diagnosed with ALS and is presented for treatment.The patient is tested to determine if the patient exhibits immunedysfunction as an immune-modulator and/or anti-apoptotic factor. If thepatient tests positive for immune dysfunction, the patient is injectedintravenously with a composition comprising a therapeutically effectiveamount of MNCs isolated from hUCBCs in combination with atherapeutically effective amount of umbilical cord blood plasma preparedas described below. Improvement in motor function is shown aftertreatment with both cord blood plasma as well as hUCBCs. Additionaladministrations of the therapeutic composition are administered withimprovement in motor function exhibited.

Human umbilical cord blood units are obtained from full-term pregnanciesby vaginal delivery and are received within 48 hours of collection.Maternal blood samples, collected as the same time as the cord blood,are tested for infectious disease markers of HIV, hepatitis B and C,syphilis, CMV and HTLV I&II. Human umbilical cord blood (hUCB) units,with maternal blood samples negative for all tested infectious markers,are processed to obtain an autologous CBP fraction and mononuclear cellpopulation as detailed below. Upon receipt, the cord blood units arediluted (1:1) with sterile phosphate buffered saline (PBS) without Mg2+or Ca2+. The cord blood is then fractionated using the density gradientsolution Ficoll (Ficoll-Paque Premium: 1.078 g/mL, Cat. No. 17544202;Millipore Sigma, St. Louis, Mo., USA) in the Sepax 2 fully automatedcell processing system (Biosafe America Inc., Houston, Tex., USA), thusallowing for the sterile collection of both CBP and MNC hUCB from eachunit of cord blood. The CBP was further centrifuged at 3000 g for 10minutes to remove any additional red blood cells. The CBP was thenaliquoted and stored at −20° C. The MNC hUCB cell numbers and viabilitywere determined using the Vi-CELL Viability Analyzer (Beckman Coulter,Brea, Calif., USA). MNC hUCB was then frozen at 5×10⁷ cells per vialusing a proprietary cryopreservation media (Saneron CCEL Therapeutics,Inc.) and stored in liquid nitrogen. Prior to administration, MNCs andCBP are thawed and combined into a therapeutic composition foradministration to the patient.

In the preceding specification, all documents, acts, or informationdisclosed do not constitute an admission that the document, act, orinformation of any combination thereof was publicly available, known tothe public, part of the general knowledge in the art, or was known to berelevant to solve any problem at the time of priority.

The disclosures of all publications cited above are expresslyincorporated herein by reference, each in its entirety, to the sameextent as if each were incorporated by reference individually.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall there between.

What is claimed is:
 1. A method of treating amyotrophic lateralsclerosis (ALS) in a patient in need thereof comprising: isolating humanumbilical cord blood cells (hUCBCs) from human umbilical cord blood;isolating a mononuclear cell fraction from the hUCBCs; isolating plasmaderived from the human umbilical cord blood; combining the mononuclearcell fraction and the plasma derived from the human umbilical cord bloodto form a composition; and administering a therapeutically effectiveamount of the composition to a patient in need thereof; wherein thecomposition comprises about 1×10⁴ to about 5×10⁷ of the mononuclearcells and about 10 ml/kg to about 20 ml/kg of the plasma derived fromthe human umbilical cord blood.
 2. The method of claim 1, wherein thehUCBCs and the umbilical cord blood plasma are autologous.
 3. The methodof claim 1, wherein multiple administrations of the umbilical cord bloodplasma are delivered to the patient within a given time period.
 4. Themethod of claim 1, wherein the umbilical cord blood plasma isnon-autologous to the patient.
 5. The method of claim 1, wherein theumbilical cord blood plasma contains increased levels of IL-8 and VEGFas compared to adult blood plasma.
 6. The method of claim 1, furthercomprising administering a therapeutic composition comprising riluzole,mesenchymal stem cells, or a combination thereof.